Injectable melphalan compositions comprising a cyclodextrin derivative and methods of making and using the same

ABSTRACT

The present invention is directed to pharmaceutical compositions comprising melphalan and a cyclodextrin derivative, and methods of making and using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 14/108,169, filed Dec. 16, 2013, which is a continuation of U.S. patent application Ser. No. 12/790,724, fled May 28, 2010, which claims the benefit U.S. Appl. No. 61/182,560, filed May 29, 2009, each of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION Field: of the Invention

The present invention relates to pharmaceutical compositions comprising melphalan and a cyclodextrin derivative and methods of making and using the same, for example, to treat disorders and diseases that are therapeutically responsive to melphalan.

Background of the Invention

Melphalan is an alkylating agent of the bischloroethylamine type, and is active against both resting and rapidly dividing tumor cells. See, e.g., U.S. Pat. Nos. 3,032,584, 3,032,585, and 4,997,651. An injectable melphalan composition (ALKERAN® for Injection, GlaxoSmithKline) has been approved by the U.S. Food and Drug Administration, for the palliative, treatment of patients with multiple myeloma for whom oral therapy is not appropriate, and an oral melphalan composition (ALKERAN® Tablets, GlaxoSmithKline) has been approved for the palliative treatment of multiple myeloma and for the palliation of non-respectable epithelial carcinoma of the ovary.

ALKERAN® for Injection (GlaxoSmithKline) is administered intravenously after first diluting a sterile, nonpyrogenic, freeze-dried powder containing melphalan hydrochloride (equivalent to 50 mg melphalan) and 20 mg povidone with a sterile diluent that contains sodium citrate (0.2 g), propylene glycol (6 mL), ethanol (96%, 0.52 mL), and water, for a total, volume of 10 mL. The usual intravenous dose is 16 mg/m², which is administered as a single infusion over 15 to 20 minutes. Melphalan is intravenously administered in 4 doses at 2-week, intervals, then, after adequate recovery from toxicity, at 4-week intervals.

According to the ALKERAN® for Injection. (GlaxoSmithKline) label, following administration of ALKERAN® for Injection, drug plasma concentrations of melphalan decline rapidly in a biexponential manner with distribution phase and terminal elimination phase half-lives of approximately 10 and 75 minutes, respectively. The average total body clearance is 7 to 9 mL/min/kg (250 to 325 mL/min/m²). A study has reported that on repeat dosing of 0.5 mg/kg every 6 weeks, the clearance of melphalan decreased from 8.1 mL/min/kg after the first course, to 5.5 mL/min/kg after the third course, but did not decrease appreciably after the third course. “Mean (±SD) peak melphalan plasma. concentrations in myeloma patients after administration of 10 or 20 mg/m² doses of melphalan were 1.2±0.4 and 2.8±1.9 μg/mL, respectively. After intravenous administration of 50 mg of melphalan, the steady-state volume of distribution of melphalan is 0.5 L/kg. The extent of melphalan binding to plasma proteins ranges from 60% to 90%. Serum albumin, is the major binding protein, while α₁-acid glycoprotein appears to account for about 20% of the plasma protein binding. Approximately 30% of the drug is (covalently) irreversibly bound to plasma proteins. Interactions with immunoglobulins have been found to be negligible.

Melphalan is eliminated, from plasma primarily by chemical hydrolysis to monohydroxymelphalan and dihydroxymelphalan. Aside from these hydrolysis products, no other melphalan metabolites have been observed in humans.

Controlled trials comparing intravenous to oral melphalan have shown greater myelosuppression with the intravenously administered melphalan. Furthermore; hypersensitivity reactions, including anaphylaxis, have occurred in approximately 2% of patients who have received intravenous melphalan. Melphalan also undergoes rapid hydrolysis in aqueous solution. See S. A. Stout et al., Int. J. Pharm. 24:193 (1985). Melphalan in the ALKERAN® For Injection (GlaxoSmithKline) product also rapidly forms a citrate derivative upon reconstitution and cannot be refrigerated, due to precipitation of melphalan from solution.

Melphalan compositions comprising a cyclodextrin derivative as a carrier and/or a diluent are known. See, e.g., Medlicott, et al., J Pharm Sci. 87:1138 (1998), D. Q. Ma et al., Int. J. Pharm. 189:227 (1999), D. Q. Ma et al., J. Pharm. Sci. 89.275 (2000), and U.S. Pat. Nos. 4,983,586, 5,024,998, and 6,583,125.

BRIEF SUMMARY OF THE INVENTION

What is needed is a melphalan formulation that can minimize the toxicology and side-effect profile of intravenous melphalan. What is also needed is an intravenous melphalan formulation having increased bioavailability and/or an improved rate of therapeutic onset. Also needed is a melphalan composition suitable for intravenous administration that is stable under ambient and/or refrigerated conditions, and can provide fully dissolved melphalan without the need for organic solubilizers (e.g., ethanol and/or propylene glycol, and the like). What is also needed is a composition free from components that rapidly form a derivative with melphalan. What is also needed is a melphalan composition suitable for intravenous administration that has improved stability, thereby enabling longer duration infusions, and lengthening the melphalan exposure time that a patient receives from a Single, convenient administration. As described herein, compositions suitable for oral or parenteral administration, that include melphalan and a cyclodextrin derivative have been developed.

The present invention is directed to a pharmaceutical composition comprising 25 mg to 125 mg of melphalan as a hydrochloride salt, an optional buffer, and a cyclodextrin derivative of formula I.

wherein n is 4, 5 or 6; wherein. R₁, R₂, R₃, R₄, R₄, R₅, R₆, R₇, R₈ and R₉ are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃ group, or an optionally substituted straight-chain or branched C₁-C₆ group; wherein, at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and is a straight-chain or branched C₁-C₈,-(alkylene)-SO₃ group; wherein the pharmaceutical composition has a pH of about 4 to about 6; wherein dilution of the pharmaceutical composition with an aqueous solution provides a melphalan solution ready for infusion in which the melphalan degrades by 2% or less at about 25° C. within 10 hours, or 4% or less at about 25° C. within 10 hours after the dilution; and wherein the cyclodextrin derivative is present in a ratio of 50:1 to 100:1 (w/w) relative to the melphalan.

The present invention is also directed to a pharmaceutical composition comprising 150 mg to 250 mg of melphalan as a hydrochloride salt, an optional buffer, and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are independently —H, a straight-chain or branched C1-C8-(alkylene)-SO₃ group, or an optionally substituted straight-chain or branched C₁-C₆ group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branched C₁-C₈-(alkylene)-SO₃ group; wherein the pharmaceutical composition has a pH of about. 4 to about 6, wherein dilution of the pharmaceutical composition with an aqueous: solution provides, a melphalan solution ready for infusion in which the melphalan degrades by 2% or less at about 25° C. within 5 hours, or 4% or less at about 25° C. within 10 hours after the dilution, and wherein the cyclodextrin derivative is present in a ratio of 25:1 to 35:1. (w/w) relative to the melphalan.

In some embodiments, the cyclodextrin derivative is a compound of formula II:

wherein. R═(H)_(21-x) or (—(CH₂)₄—SO₃—Na⁺)_(x), and x=6.0-7.1.

In some embodiments, the cyclodextrin derivative is a compound of formula II:

wherein R═(H)_(21-x) or (—(CH₂)₄—SO₃—Na⁺)_(x), and x=6.0-7.1; and

and the pharmaceutical composition comprises about 50 mg of melphalan as a hydrochloride salt and the cyclodextrin derivative is present in a concentration of 50:1 to 100:1 (w/w) relative to the melphalan; or

the pharmaceutical composition comprises about 50 mg of melphalan as a hydrochloride salt the cyclodextrin derivative is present in a ratio of about 55:1 (w/w) relative to the melphalan; or

the pharmaceutical composition comprises about 200 mg of melphalan as a hydrochloride salt and the cyclodextrin derivative is present in a ratio of 25:1 to 35:1 (w/w): relative to the melphalan; or

the pharmaceutical composition comprises about 200 mg of melphalan as a hydrochloride salt and the cyclodextrin derivative is present in a ratio of about 27:1, about 30:1, or about 32:1 (w/w) relative to the melphalan.

The present invention is directed to a method of treating a subject suffering from a neoplastic disorder, the method comprising diluting a composition with an aqueous diluent to provide a dilute pharmaceutical composition comprising 25 mg to 125 mg of melphalan and a cyclodextrin derivative of formula 1:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃ group, or an optionally substituted straight-chain or branched C₁-C₆ group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branched. C₁-C₈-(alkylene)-SO₃ group; wherein the dilute pharmaceutical composition has a pH of about 4 to about 6; wherein the cyclodextrin derivative is present in a concentration of at least 50:1 (w/w) relative to the melphalan; wherein the melphalan in the dilute pharmaceutical composition degrades by 2% or less at about 25° C. within 5 hours, or 4% or less at about 25° C. within 10 hours after the diluting; and administering the dilute pharmaceutical composition by injection to the subject in need thereof.

In some embodiments, the neoplastic disorder is selected from: myeloma, multiple myeloma, acute myelogenous leukemia, melanoma, malignant melanoma, breast cancer, ovarian cancer, testicular cancer, advanced prostate cancer, a neuroendocrine cancer, metastatic melanoma (e.g., metastatic ocular melanoma, metastatic cutaneous melanoma, and the like), a metastatic neuroendocrine tumor, a metastatic adenocarcinoma tumor, hepatocellular carcinoma, osteogenic sarcoma, polycythemia veraplasma, plasma cell neoplasm, amyloidosis, sclerormyxedema, and combinations thereof. In some embodiments, the neoplastic disorder is multiple myeloma and the administering is systemic and provides palliative treatment of the multiple myeloma.

The present invention is also directed. to a method for conditioning a subject in need of a stem cell transplantation, the method, comprising administering a melphalan dose of 50 mg/m² to 300 mg/m² per clay to the subject in need of the stem cell transplantation, wherein the melphalan dose is administered in a pharmaceutical composition comprising melphalan and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃ group, or an optionally substituted straight-chain or branched C₁-C₆ group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branched. C₁-C₈-(alkylene)-SO₃ group; wherein the pharmaceutical composition has a pH of about 4 to about 6; and wherein the cyclodextrin derivative is present in a ratio of at least 25:1 (w/w) relative to the melphalan.

In some embodiments, at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R is a hydroxy-substituted-C₃ group.

In some embodiments, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are independently a straight-chain, or branched C₁-C₈-(alkylene)-SO₃— group having a degree of substitution of 4 to 8 per cyclodextrin derivative, and the remaining substituents are —H.

In some embodiments, at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is substituted with a straight-chain C₄-(alkylene)-SO₃— group.

In some embodiments, a pharmaceutical composition or a dilute pharmaceutical composition is substantially free of an alcohol.

In some embodiments, the aqueous diluent is a saline solution.

In some embodiments, a. dilute pharmaceutical composition is stored about 0.5 hours to about 48 hours prior to the administering. In some embodiments, the melphalan in a pharmaceutical composition of the present invention degrades by 2% or less at about 25° C. within 5 hours, or 4% or less at about 25° C. within 10 hours after the diluting.

In some embodiments, a subject suffering from a neoplastic disorder or in need of a stem cell transplantation is a pediatric subject.

In some embodiments, the administering is performed intravenously. In some embodiments, the administering is performed via a limb perfusion.

In some embodiments, the administering is for a period of two or more days.

In some embodiments, the administering provides a melphalan C_(max) in a subject that is at least 20% or greater than a melphalan C_(max) provided by a melphalan formulation containing, an equivalent dose of melphalan and lacking the cyclodextrin derivative. In some embodiments, the administering provides a melphalan AUC₀₋₁ in a subject that is at least 20% or greater than a melphalan AUC₀₋₁ provided by a melphalan formulation containing an equivalent dose of melphalan and lacking the cyclodextrin derivative.

In some embodiments, a method of the present invention comprises diluting a concentrated melphalan composition with an aqueous diluent to provide the pharmaceutical composition. In some embodiments, a concentrated melphalan composition, comprises 50 mg to 500 mg melphalan. In some embodiments, a concentrated melphalan composition comprises about 200 mg of melphalan.

The present invention is also directed to a pharmaceutical kit comprising a first container comprising 25 mg to 125 mg of melphalan as a hydrochloride salt and an optional water-soluble polymer, and a second container comprising an aqueous diluent, an optional buffer, and a cyclodextrin derivative of formula I.

wherein la is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are independently —H, as Straight-chain or branched C₁-C₈-(alkylene)-SO₃ group, or an optionally. substituted straight-chain or branched C₁-C₆ group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branched C₁-C₈-(alkylene)-SO₃ group; wherein the cyclodextrin derivative is present in the second container in a concentration of at least 50.1 (w/w) relative to the melphalan; and wherein combining the first container and the second container provides a dilute pharmaceutical composition having a pH of about 4 to about 6 that degrades by 2% or less at about 25° C. within 5 hours, or 4% or less at about 25° C. within 10 hours after the diluting.

The present invention is also directed to a. pharmaceutical kit comprising a first container comprising 150 mg to 250 mg of melphalan as a hydrochloride salt and an optional water-soluble polymer; and a second container comprising an aqueous diluent; an optional buffer, and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; Wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃ group, or an optionally substituted straight-chain or branched C₁-C₆ group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branched C₁-C₈-(alkylene)-SO₃ group; wherein the cyclodextrin derivative is present in the second container in a Concentration of 25:1 to 35:1 (w/w) relative to the melphalan; and wherein combining the first container and the second container provides a dilute pharmaceutical composition having a pH of about 4 to about 6 that degrades by 2% or less at about 25° C. within 5 hours, or 4% or less at about 25° C. within 10 hours after the diluting.

In stone embodiments, a first container comprises povidone in an amount of 10 mg to 30 mg, and a second container comprises a pH-adjusting agent in a concentration sufficient to provide a pH of about 4 to about 6 when, the. first container and the second container are combined.

In some embodiments, the cyclodextrin derivative present in. the second container is a compound of formula II:

wherein. R═(H)_(21-x) or (—(CH₂)₄—SO₃—Na⁺)_(x), and x=6.0-7.1; wherein the first container comprises about 200 mg of melphalan as a hydrochloride salt; and, wherein the cyclodextrin derivative is present in the second container in an amount of about 27:1, about 30:1, or about 32:1 (w/w) relative to the melphalan.

Further embodiments, features, and advantages of the present inventions, as well as the composition, structure and operation of the various embodiments of the present invention, are described in detail below with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying drawings, which are incorporated, herein and form a part of the specification, illustrate one or more embodiments of the present invention and, together with the description, further serve to explain the principles, of the invention and to enable a person skilled in the pertinent art to make and use the invention.

FIG. 1 provides a graphic representation of the solubility of free base melphalan as a function of pH and the concentration of a. cyclodextrin derivative.

FIG. 2 provides a graphic representation of the solubility of free base melphalan and melphalan hydrochloride at pH 7.5 as a function of the concentration of a cyclodextrin derivative.

FIGS. 3. and 4 provide flow charts that describe processes for preparing a unit dosage form of the present invention.

FIGS. 5A-5B provide a graphic representation of dose-normalized whole. blood (FIG. 5A) and plasma (FIG. 5B) concentrations of melphalan following intravenous administration to male Sprague-Dawley rats using a melphalan formulation containing a Cyclodextrin derivative (SBE_(6:5)-β-CD) and a cyclodextrin-free melphalan formulation (ALKERAN® for Injection, GlaxoSmithKline).

FIG. 6 provides a graphic representation of the mean plasma melphalan concentration in a human patient after intravenous administration of a melphalan formulation containing a cyclodextrin derivative (SBE_(6.5) β-CD) and after intravenous administration of a cyclodextrin-free melphalan formulation (Melphalan HCl Injectable, Bioniche Pharma USA).

One or more embodiments of the present invention will now be described with reference to the accompanying drawings. In the drawings, like reference numbers can indicate identical or functionally similar elements. Additionally; the left-most digit(s) of a reference number can identify the drawing in which the reference number first: appears.

DETAILED DESCRIPTION OF THE INVENTION

This specification discloses one or more embodiments that incorporate the features of this invention. The disclosed embodiment(s) merely exemplify the invention. The scope of the invention is not limited to the disclosed embodiment(s). The invention is. defined by the claims appended hereto.

Throughout the specification, use of the term “about” with respect to any quantity is contemplated to include that quantity: For example; “about 10 mL” is contemplated herein to include “10 mL,” as well, as values understood in the art to be approximately 10 mL. with respect to the entity described.

The invention includes combinations and sub-combinations of the various aspects and embodiments disclosed herein. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is understood that it is within the knowledge of one skilled in the art to effect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described. These and other aspects of this invention will be apparent upon reference to the following detailed description, examples, claims and attached figures.

Melphalan

The compositions, formulations and unit dosage forms of the present invention comprise melphalan, which has the following chemical structure:

As used herein, the term “melphalan” refers to the L-isomer of the above compound, 4-[bis(chloroethyl)amino]phenylalanine, as well as addition salt, polymorphs, solvates, hydrates, dehydrates, co-crystals, anhydrous, and amorphous forms thereof. Melphalan contains a chiral atom, and thus, as used herein, “melphalan” can refer to the substantially pure form of the L-isomer. As used herein, “substantially pure” refers to melphalan having a purity of 90% or higher, 95% or higher, 98% or higher, 99% or higher, 99.5% or higher, or 99.9% or higher.

The D-isomer of the above compound, known as melphalan, is less active against certain animal tumors, and the dose needed to produce effects on chromosomes is larger than that required with melphalan. The racemic (DL-) form is known as melphalan or sarcolysin. In some embodiments, the compositions of the present invention are substantially free of melphalan. In some embodiments, the compositions of the present invention comprise melphalan as a hydrochloride salt having a purity of 95% or greater, 98% or greater, 99% or greater, 99.9% or greater, or 99.99% or greater.

Melphalan is a bifunctional alkylating agent that is active against selected human neoplastic diseases. The molecular formula for melphalan is C₁₃H₁₈Cl₂N₂O₂, and the molecular weight of the free base form is 305.20 g/mol. Melphalan is practically insoluble in water (pH 7) and has a pKa, of about 2.5.

In some embodiments, the pharmaceutical compositions and dosage forms of the present invention comprise melphalan as a hydrochloride salt. As used herein, “melphalan as a hydrochloride salt” refers to the hydrochloric acid addition salt of the above compound. However, amounts and concentrations of melphalan are provided in reference to an, equivalent mass of free base melphalan. Thus, 5 mg of “melphalan as a hydrochloride salt” refers to 5 mg of the active agent melphalan, exclusive, of the hydrochloride addition salt, which: if considered would provide a total mass of about 5.6 mg.

Cyclodextrin Derivatives

The compositions, formulations and/or unit dosage forms of the present invention comprise a cyclodextrin derivative. As used herein, “cyclodextrin derivative” refers to a cyclic oligosaccharide comprising five or more α-D-glucopyranoside units linked in, circular 1→4 configuration, and comprising a substituent group attached to one or more of the glucopyranoside units at the 2, 3 and/or 6 position(s) through an ether bond (O—R—, where R refers to the substituent group).

In some embodiments, the cyclodextrin derivative s a compound of formula I:

wherein: n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are independently selected, from: —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃ group, and an optionally substituted straight-chain, or branched. C₁-C₆ group.

In some embodiments, cyclodextrin for use with the present invention is selected based, upon an average degree of substitution (“ADS”), which, as used herein refers to the average number of substituent groups per cyclodextrin molecule. The average degree of substitution for cyclodextrin derivatives is described in detail in WO 2009/018069, which is incorporated, herein by reference in its entirety. As used herein, a cyclodextrin derivative composition for use with the present invention is referred to by the following notation: the substituent(s) are abbreviated (e.g., sulfobutyl ether groups are abbreviated as “SBE”) with a subscript denoting the ADS of the substituent, and cyclodextrin structure is defined. For example, a sulfobutyl ether-derivatized β-cyclodextrin composition having an ADS of 6.5 is referred to as “SBE_(6.5)-β-CD.” As a second example, a β-cyclodextrin composition comprising cyclodextrin molecules derivatized with both sulfobutyl ether and hydroxypropyl groups is referred to as “SBE_(4.2)-HP_(2.5)-β-CD,” wherein the ADS of the sulfobutyl ether groups is 4.2 and the ADS of the hydroxypropyl groups is 2.5.

Cyclodextrin derivatives suitable for use with the present invention include cyclodextrin compositions bearing substituent groups (R₁-R₉ and R in formulas I and II, respectively) that are independently selected from: —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃ group, and an optionally substituted straight-chain or branched C₁-C₆ group.

In some embodiments, at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ of formula I is substituted with a straight-chain C₄-(alkylene)-SO₃ group. Exemplary C₁-C₈-(alkylene)-SO₃ groups suitable for use with the present invention include, but are not limited to, sulfoethyl, sulfopropyl, 1-methyl-sulfopropyl, sulfobutyl, 1-methyl-sulfobutyl, 2-methyl-sulfobutyl, 1-methyl-sulfobut-3-yl, 2-ethyl-sulfobutyl, 3-ethyl-sulfobutyl, sulfopentyl, 1-sulfopent-3-yl, sulfohexyl, sulfoheptyl, sulfooctyk and the like, and combinations thereof.

In some embodiments, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ of formula I are independently a straight-chain or branched C₁-C₈-(alkylene)-SO₃ group having an ADS of 4 to 8, 4 to 7.5, 4 to 7, 4 to 6.5, 4.5 to 8, 4.5 to 7.5, 4.5 to 7, 5 to 8, 5 to 7.5, 5 to 7, 5.5 to 8, 5.5 to 7.5, 5.5 to 7, 5.5 to 6.5, 6 to 8, 6 7.5, 6 to 7.1, 6.5 to 7, about 6.5, or about 7 per cyclodextrin derivative, and the remaining substituents are —H.

In some embodiments, a substituent is an optionally substituted straight-chain or branched C₁-C₆ group. As used herein, “optionally substituted” refers to one or more optional substituents selected from: halogen (i.e., —F, —Cl, —Br, —I), —NO₂, —C═N, —OR₂₂, —SR₂₂, —SO₂R₂₂, —C(═O)OR₂₂, —C(═O)R₂₂, —C(═O)N(R₂₂)₂, —SO₂N(R₂₂)₂, —SO₂N(H)C(═O)R₂₂, —SO₂N(H)C(═O)OR₂₂ (wherein R₂₂ is not H), —N(R₂₂)₂, —N(R₂₂)SO₂R₂₂, —N(R₂₂)C(O)_(m)R₂₂ (wherein m=1 or 2), —N(R₂₂)C(O)N(R₂₂)₂, —N(R₂₂)SO₂N(R₂₂)₂, —O—C(═O)R₂₂, —O—C(═O)OR₂₂, —O—C(═O)N(R₂₂)₂, —C(═O)N(H)SO₂N(R₂₂)₂, —C(═O)N(H)SO₂R₂₂, oxo (or keto, i.e., ═O); thioxo (i.e., ═S), imino (i.e., ═N(R₂₂), —NR₂₂—C(═NR₂₂)R₂₂, —NR₂₂—C(═NR₂₂)N(R₂₂)₂, —C(═NR₂₂)N(R₂₂)₂, —O—C(═NR₂₂)N(R₂₂)₂, —O—C(═NR₂₂)R₂₂, —C(═NR₂₂)R₂₂, —C(═NR₂₂)OR₂₂, and ionic forms thereof (e.g., —N⁺(R₂₂)₂X⁻, and the like, wherein X— is a pharmaceutically acceptable anion), wherein R₂₂ is independently selected at each occurrence from H, and C₁-C₄ alkyl.

Exemplary optionally substituted straight-chain or branched C₁-C₆ groups include, but are not limited to, 2-hydroxypropyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 3-oxobutyl, and 2-ethoxy-ethyl.

In some embodiments, at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ of formula I is a hydroxy-substituted-C₃ group. In some embodiments, the cyclodextrin derivative comprises β-cyclodextrin that includes, a hydroxy-substituted-C₃ group having an ADS of 1 to 8, 2 to 8, 3 to 7, 4 to 7.5, 4.3 to 7.5, about 1, about 2, about 2.5, about 3, about 3.5, about 4, about 4.3, about 5, about 5.5, about 6, about 6.5, about 7, or about 7.5.

Exemplary cyclodextrin compositions, and methods of making the same, that are suitable for use with the present invention also include those described in U.S. Pat. Nos. 5,134,127, 5,241,059, 5,376,645, 5,874,418, 6,046,177, 6,133,248, 6,153,746, 6,204,256, 7,034,013, 7,629,331, and 7,635,773, U.S. Pub. No. 2009/0012042, and PCT Pub. No. WO 2005/117911, the contents of each of which is incorporated herein by reference, in the entirety.

In some embodiments, the cyclodextrin derivative is a compound of formula II.

wherein R(H)_(21-x) or (—CH₂)₄—SO₃Na⁺)_(x). In some embodiments, x=6.0-7.1. In some embodiments, the cyclodextrin derivative of formula II has an average molecular weight of about 2163 g/mol.

In some embodiments, the cyclodextrin derivative is a sulfobutyl ether-β-cyclodextrin having an ADS of about 7 (e.g., CAPTISOL®, CyDex Pharmaceuticals, Inc., Lenexa, Kans.). Captisol, cyclodextrin is a polyanionic β-cyclodextrin derivative with a sodium sulfonate salt separated from the lipophilic cyclodextrin cavity by a butyl ether spacer group, or sulfobutylether (SBE). CAPTISOL® cyclodextrin has been shown to be safe when administered parenterally, orally, or via inhalation and does not exhibit the nephrotoxicity associated with. β-cyclodextrin. Relative, to β-cyclodextrin, CAPTISOL® sulfoalkyl ether cyclodextrin provides comparable or higher complexation characteristics and superior water solubility in excess of 90 g per 100 mL, a 50-fold improvement. Melphalan has a low binding affinity with CAPTISOL® (K_(a)=3×10² M¹).

In some embodiments, the cyclodextrin derivative includes a substituent that, bears an ionic group that can optionally form a salt with a pharmaceutically acceptable anion or cation. Pharmaceutically acceptable cations suitable for forming salts with. negatively charged cyclodextrin derivatives of the present invention include, but are not limited to, H⁺Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, ammonium and amine cations such as cations of (C₁-C₆) alkylamines, (C₄-C₈)-cycloalkylamines (e.g., piperidine, pyrazine, and the like), (C₁-C₆) alkanolamines, and (C₄-C₈)-cycloalkanolamines, and the like; and combinations thereof. In some embodiments, a pharmaceutically acceptable cation is Na⁺. Pharmaceutically acceptable anions suitable for forming salts with positively charged cyclodextrin derivatives of the present invention include, but are not limited to, halides (e.g., Cl⁻ and the like), anions of (C₁-C₆)-alkyl acids (e.g., acetate, oxalate, fumarate, succinate, and the like, and combinations thereof.

Pharmaceutical Compositions and Unit Dosage Forms

The present invention is directed to pharmaceutical compositions and unit dosage forms comprising, melphalan and a cyclodextrin derivative. The pharmaceutical compositions of the present invention are suitable for parenteral administration to a subject. Parenteral administration of the pharmaceutical compositions can include, but is not limited to, an injection. Because parenteral administration bypasses a subjects natural defenses against contaminants, the pharmaceutical compositions are sterile, or capable of being sterilized prior to administration.

Exemplary pharmaceutical compositions include, but are not limited to, solutions, suspensions or emulsions ready for administration, solutions, suspensions or emulsions ready to be dissolved in and/or diluted with a pharmaceutically acceptable vehicle, and dry products ready to be dissolved in and/or diluted with a pharmaceutically: acceptable vehicle:

Generally, the pharmaceutical compositions of the present invention comprise melphalan in a concentration suitable for treating a condition that is amenable: to treatment with melphalan. Thus, the pharmaceutical compositions of the present invention can be used to prepare a unit dosage form comprising: a therapeutically effective amount of melphalan for administering to a subject in need thereof. In some embodiments, the present invention is directed to a unit dosage form that comprises melphalan in a concentration that is suitable for administration without dilution. Alternatively, a unit dosage form of the present invention, can be diluted prior to administration to a subject in need thereof.

The present invention is also directed to: a pharmaceutical composition comprising 25 mg to 125 mg, 25 mg to 100 mg, 25. mg to 75 mg, 25 mg to 50 mg, 50 mg to 125 mg, 50 mg to 100 mg, 75 to 125 mg, 100 to 125 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, or about 125 mg of melphalan as a hydrochloride salt, an optional buffer, and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃ group, or an optionally substituted straight-chain or branched C₁-C₆ group; wherein, at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branched C₁-C₈-(alkylene)-SO₃ group; wherein the pharmaceutical composition has a pH of about 4 to. about 6, about 4 to about 5, about 4.5 to about 6, about 5 to about 6, about 5.5 to about 6, about 4, about 4.5, about 5, about 5:5, or about 6; wherein dilution of the pharmaceutical composition with an aqueous solution provides a solution in which the melphalan degrades by 2% or less at about 25° C. within 5 hours, or by 4% or less at about 25° C. within 10 hours after the dilution; and wherein the cyclodextrin derivative is present in a ratio: of 50:1 to 100:1, 55:1 to 60:1, about 50:1, about 55:1, or about 60:1 (w/w) relative to the melphalan.

The present: invention is also directed to. a pharmaceutical composition comprising 150 mg to 300 mg, 150 mg to 250 mg, 150 mg to 225 mg, 175 mg to 250 mg, 200 mg to 250 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, or about 250 mg of melphalan as a hydrochloride salt, an optional buffer, and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃ group, or an optionally substituted straight-chain or branched C₁-C₆ group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇; R₈ and R₉ is a straight-chain or branched C₁-C₈-(alkylene)-SO₃ group; wherein the pharmaceutical composition has a pH of about 4 to about 6, wherein dilution of the pharmaceutical composition with a aqueous solution provides a melphalan solution in Which the melphalan degrades by 2% or less at about 25° C. within 5 hours, or 4% or less at about 25° C. within 10 hours after the dilution; and wherein the cyclodextrin. derivative is present in. a ratio of 25:1 to 35:1, about 0.27:1, about 30:1, or about 32:1 (w/w) relative to the melphalan.

In some embodiments, the cyclodextrin derivative is a compound of formula II:

wherein R═(H)_(21-x) or (—(CH₂)₄—SO₃—Na)_(x), and x=6.0-7.1,

In some embodiments, the cyclodextrin derivative is a compound of formula II:

wherein R═(H)_(21-x) or (CH₂)₄—SO₃—Na⁺)_(x), and x=6.0-7.1; and

the pharmaceutical composition comprises about 50 mg of melphalan as a hydrochloride:salt and the cyclodextrin derivative is present in a concentration of 50:1 to 100:1, 55:1 to 60:1, about 50:1, about 55:1, or about 60:1 (w/w relative to the melphalan; or

the pharmaceutical. composition: Comprises about 200 mg of melphalan as a hydrochloride salt and the cyclodextrin derivative is present in a ratio of 25:1 to 35:1, about 27:1, about 30:1, or about 32:1 (w/w) relative to the melphalan.

Sterile solutions, suspensions, emulsions and the like can be prepared by incorporating melphalan into an appropriate solvent or carrier with the other, optional ingredients enumerated herein, followed by sterilization. Sterile powders can be prepared by spray drying, aseptic spray drying, vacuum drying, or freeze drying a sterile, solution, suspension, or emulsion to′ provide a dried solid (e.g., a powder) comprising melphalan along with any additional excipients.

In some embodiments, the present invention is directed to a′ solid pharmaceutical composition consisting of about 50 mg of melphalan as a hydrochloride salt, an amount sufficient of an acid, a. base, or a combination thereof to provide, a pH of about 4 to about 6 upon dilution with a saline solution to a volume of about 10 mL, and a cyclodextrin derivative of formula II:

wherein R═(H)_(21-x) or (—(CH₂)₄—SO₃—Na⁺)_(x), and x=6.0-7.1, wherein dilution of the solid pharmaceutical composition with an aqueous solution provides a melphalan solution in which the melphalan degrades by 2% or less at about 25° C. within 5 hours, or by 4% or less at about 25° C. within 10 hours after the diluting, and wherein the cyclodextrin derivative is present in a ratio of about 55:1 (w/w) relative to the melphalan.

In some embodiments, the present invention is directed to a solid pharmaceutical composition consisting of about 200 mg of melphalan as a hydrochloride: salt, an amount sufficient of an acid, a base, or a combination thereof to: provide a pH of about 4 to about 6 upon dilution with a saline: solution to a volume of abut. 20 mL, and a cyclodextrin derivative of formula II:

wherein R═(H)_(21-x) or (—(CH₂)₄—SO₃—Na⁺)_(x), and x=6.0-7.1, wherein dilution of the solid pharmaceutical composition with an aqueous solution provides a melphalan solution in which the melphalan degrades by 2% or less at about 25° C. within 5 hours, or by 4% or less at about 25° C. within 10 hours after the diluting, and wherein the cyclodextrin derivative is present in a ratio: of about 27:1, about 30:1, or about 32:1 (w/w) relative to the melphalan.

In some embodiments, a pharmaceutical composition: or unit dosage form of the present invention comprises a solid (e.g., a powder) or a liquid solution that is diluted with a liquid carrier or diluent prior to administration to a subject. Thus, the pharmaceutical compositions: and unit dosage forms of the present invention include sterile aqueous solutions, suspensions and dispersions, as well as sterile solids (e.g., powders) comprising melphalan that can be extemporaneously diluted or solubilized to provide a sterile solution, suspension or dispersion.

In some embodiments, the compositions, formulations and/or unit dosage forms of the present invention comprise a pharmaceutically acceptable excipient. As used herein, “pharmaceutically acceptable” refers to those excipients, compounds, materials, and/or compositions which are, within the: scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other possible complications commensurate with a reasonable benefit/risk ratio.

In some embodiments, the: pharmaceutical compositions and unit dosage forms of the present invention are substantially homogeneous. As used herein, “homogeneous” refers to mixtures, solutions, suspensions, compositions, dosage forms, and/or formulations of the present invention that have a uniform distribution of ingredients throughout. Homogeneity is synonymous with uniformity and can refer to intra-sample uniformity, batch-to-batch uniformity, run-to-run uniformity, and/or dosage form-to-dosage form uniformity. For example, intra-sample uniformity can be determined by analyzing a first: portion of a sample,: Mixture, or composition and comparing this with a second portion of the same sample, mixture, or composition. Typical deviations of a composition (e.g., variation in the percentage by weight of excipients: and the like) of a substantially homogeneous composition are about 5% or less, about 3% or less, about 2% or less; about 1% or less, or within experimental error.

In some embodiments, a pharmaceutical composition or unit dosage form of the present invention comprises a pharmaceutically acceptable excipient. As: used herein, the term “excipient” refers to any inert. substance that can, be combined with melphalan and the sulfoalkyl ether cyclodextrin for preparing the pharmaceutical compositions.

Pharmaceutically acceptable excipients suitable for use with the present. invention include, but are not limited to, a carrier; a water-soluble polymer, a preservative, an antioxidant, a pH-adjusting agent (e.g., an acidifying, agent, an alkalinizing agent, and/or a buffer), a bulking. agent, a complexation enhancing agent, a cryoprotectant, a density modifier, an electrolyte, a flavor, a fragrance, a lyophilizing aid (e.g., a bulking agent and/or stabilizing agent), a plasticizer, a solubility-enhancing agent, a stabilizing, agent, a sweetener, a surface tension modifier, a volatility modifier, a viscosity modifier, and combinations thereof. In addition, one of skill in the art will recognize that pharmaceutically acceptable excipients can be used in the present invention, including those listed in The Handbook of Pharmaceutical Excipients, 5th Ed., The Pharmaceutical Press and American Pharmacists Association, London, U K and Washington, D.C. (2006), which is incorporated herein by reference in its entirety.

In some embodiments, a pharmaceutical composition or unit dosage form of the present invention comprises a pharmaceutically acceptable carrier. As used herein, a “carrier” refers to a vehicle suitable for transferring and/or diluting a pharmaceutical composition or unit dosage form of the present invention. Pharmaceutically acceptable carriers suitable for use with the present Inventions include, but are not limited to, liquids, solids, colloids, gels, and combinations thereof. Liquid carriers suitable for use with the present invention include solvents, liquid dispersion mediums, and the like, such as, but not limited to, water, ethanol, a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycols, and the like), a vegetable, oil, a nontoxic glyceryl ester, and combinations thereof. In some embodiments, a liquid carrier is selected from a dextrose solution, a saline solution, plasma, and lactated Ringer's solution.

In some embodiments, a pharmaceutical composition or unit dosage form of the present invention comprises a water-soluble polymer such as, but not limited to, homopolymers of N-polyvinylpyrrolidone (e.g., “povidone”), low molecular weight hydroxypropyl cellulose, low molecular weight methyl cellulose, low molecular weight hydroxypropyl methyl cellulose, and the like, and combinations thereof.

In some embodiments, after dilution a cyclodextrin derivative is present in the diluted pharmaceutical composition in a concentration of about 75 mM, about 100 mM, or about 125 mM.

In some embodiments, after dilution melphalan is present in the diluted pharmaceutical composition in a: concentration of 0.1 mg/mL to 50 mg/mL, 0.15 mg/mL to 40 mg/mL, 0.2 mg/mL to 30 mg/mL, 0.3 mg/mL to 25 mg/mL, 0.4 mg/mL to 20 mg/mL, 0.45 mg/mL to 15 mg/mL, 0.5 mg/mL to 10 mg/mL, about 0.45 mg/mL, about 1 mg/mL, about 1.5 mg/mL, about 2 mg/mL, about 2.5 mg/mL, or about 5 mg/mL.

In some embodiments, a pharmaceutical composition and/or a diluent for use with a composition of the present invention, is free of a solubilizing agent such as, but not limited to, water, an alcohol (e.g., ethanol and the like), a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycols, and the like), a vegetable oil, a nontoxic glyceryl ester, and combinations thereof. Thus, in some embodiments the diluent consists essentially of water and optional. tonicity-adjusting agents (e.g., 0.9% saline solution for injection, and the like).

In some embodiments, the pH of a pharmaceutical composition or unit. Dosage form is controlled. In some embodiments, a pharmaceutical composition or unit dosage form of the present. invention comprises a pharmaceutically acceptable buffer and/or pH. adjusting agent (e.g., an. acidifying agent. and/or alkalinizing agent). In some embodiments, a pharmaceutical composition or unit dosage. form of the present invention has a pH of about 4 to about 6, about 4 to about 5, about 5 to about 6, about 4, about 5, about 5.5, or about 6 after dilution with an aqueous diluent.

In some embodiments, a pharmaceutical composition or unit dosage form that is to be diluted prior to administration to a subject has a pH of about 2 to about 6, about 3 to about 6, about 4 to about 6, or about 5 to about 6. In some embodiments, after dilution with an liquid carrier) a unit: dosage form of the present invention has a pH of about 4 to about 6, about 4 to about 5, about 5 to about 6, about 4, about 4.5, about 5, about 5.5, or about 6 at the time of administration to a subject in need thereof.

In some embodiments, a pharmaceutical composition or unit dosage form of the present invention comprises a buffer. In some embodiments; a pharmaceutical composition or unit dosage form of the present invention comprises a buffer suitable to provide a dilute composition having a pH of about 4 to about 6, about 4 to about 5, about 5 to about 6, about 4, about 4.5, about 5 about 5.5, or about 6. In some embodiments, a buffer is present in a concentration of about 0.01 M to about 10 M, about 0.01 M to about 5 M, or about 0.01 M to about 1 M.

In some embodiments, a pharmaceutical composition or unit dosage form of the present: invention comprises a pH-adjusting agent such as, but not limited to, an acidifying agent (e.g., citric acid, HCl, and the like), an alkalinizing agent (e.g., NaOH and the like), a salt form of an acid (e.g., sodium citrate and the like), and combinations thereof. In some embodiments, a pharmaceutical composition or unit dosage form of the: present invention comprises a pH-adjusting agent in an amount sufficient to provide dilute composition having a pH of about 4 to about 6, about 4 to about 5, about 5 to about 6, about 4, about 4.5, about 5 about. 5.5, or about 6. In some embodiments, In some embodiments, a pharmaceutical composition or unit dosage form, of the present invention comprises sodium citrate in an amount of 50 mg to 500 mg, 75 mg to 400 mg, 100 mg to 300 mg, 150 mg to 250 mg, or about 200 mg.

In some embodiments, a pharmaceutical composition or unit dosage form of the present, invention comprises a second therapeutic agent. Suitable second therapeutic agents include, but are not limited to, a platinum compound, an antimetabolite, a nitrosourea, a corticosteroid, a calcineurin inhibitor, a monoclonal antibody, a polyclonal antibody, a cytotoxic antibiotic, an interferon, an opioid, an antihistamine, a volume expander, a pressor agent, and combinations thereof. Additional second therapeutic agents include, but are not limited to, doxorubicin, bortezomib, rituximab, thalidomide, lenalidomide, gemcitabine, thiotepa, fludarabine, carmustine, etoposide, cytarabine, granulocyte colony-stimulating factor, ADH-1, topotecan, palifermin, prednisone, arsenic trioxide, ascorbic acid, busulfan, buthionine sulfoximine, and combinations thereof.

As used herein, a “unit dosage form” refers to a composition containing a specific amount of melphalan, the whole of which is intended, to be administered to a subject in a single dose: A unit dosage form: can be distinguished from a supply of a multi-dose amount of a pharmaceutical composition, e.g., a bottle of medicine, from which a unit dose is measured out.

In some embodiments, a. unit dosage form of the present invention comprises a therapeutically effective amount of melphalan. As used. herein, a “therapeutically effective amount” refers to an amount of melphalan that, elicits a. biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of a disease or disorder being treated.

A unit dosage form typically comprises the pharmaceutical composition of the present invention and optionally, one or more pharmaceutically acceptable excipients, wherein the amount of melphalan present in the unit dosage form is sufficient for a single administration to. a subject in. need thereof. Unit dosage forms of the present invention include, but are not limited to, liquid solution, liquid suspensions, liquid dispersions, emulsions, gels, powders, tablets, capsules, caplets, and the like. Treatment of a disease or condition amenable to treatment with melphalan can comprises periodic administration of a unit dosage form of the present invention, for example, once every two weeks, once every four weeks, or some, other interval.

In some embodiments, a unit dosage form of the present invention comprises 25 mg to 125 mg, or 150 mg to 250 mg of melphalan as a hydrochloride salt. In some embodiments, a unit dosage form of the present invention comprises 50 mg or 200 mg melphalan as a hydrochloride Salt.

In some embodiments, a unit dosage form of the present invention is a solid. In some embodiments, a solid unit, dosage form of the present invention is a lyophilized solid, or an aseptic spray-dried solid. In some embodiments, a dosage form of the present invention is suitable for dilution and/or reconstitution with a predetermined amount of a liquid carrier: For example, a unit dosage form (e.g., as liquid or a solid) of the present invention can be diluted with 5 mL to 500 mL, 10 mL to 100 mL, or 10 mL, to 50 mL of a liquid carrier.

The pharmaceutical compositions: and. unit dosage forms of the present invention are stable. As used herein, stability can refer to. either the shelf-life of an undiluted solid or liquid dosage form or the resistance to degradation of a diluted liquid dosage form. In particular; currently available melphalan compositions suitable for intravenous administration must be used as soon as possible after dilution due to the rapid degradation of melphalan in aqueous solution. However, the dosage forms of the present invention are stable for a considerable time period after dilution, for example; at least 90 minutes up to at least 48 hours or more. Thus, in those embodiments in which a solid or liquid unit dosage form is diluted, the diluting can be performed immediately prior to administering, or sometime before the administering without any significant loss of therapeutic efficacy. This enables a liquid pharmaceutical composition or liquid unit dosage form of the present invention to be diluted 90 minutes to 48 hours in advance of use (i.e., in advance of parenteral administration to a subject in need thereof).

In some embodiments, the melphalan in a pharmaceutical composition of the present invention degrades by 2% or less at about 25° C. within 5 hours, or by 4% or less at about 25° C. within 10 hours after dilution with an aqueous diluent to provide a diluted composition comprising a cyclodextrin derivative in a concentration of about 75 mM or about 125 mM.

The primary degradation, product of melphalan in aqueous solution is melphalan monohydroxide (also known as monohydroxyrnelphalan), which proceeds via a hydrolysis reaction. See, e.g., S. A. Stout et al., Int Pharm. 24:1.93 (1985). In some embodiments, dilution of a pharmaceutical composition of the present invention provides a melphalan monohydroxide concentration (based on a 100% initial concentration. of melphalan) of 2% or less within 5 hours of the diluting, when the diluted composition is maintained at room temperature (about 25° C.). In some embodiments, dilution of a pharmaceutical composition of the present invention provides a melphalan monohydroxide concentration (based on 100% initial concentration of melphalan) of 4% or less within 10 hours of the diluting, when the diluted composition is maintained at room temperature (about 25° C.). In some embodiments, dilution of a pharmaceutical composition of the. present invention provides a melphalan monohydroxide concentration (based on a 100% initial concentration of melphalan) of 2% or less within 24 hours of the diluting, or 4% or less within 48 hours of the diluting when the diluted composition is maintained at a, temperature of about 10° C. or less.

Furthermore, the Pharmaceutical compositions of the present invention can be stored prior to dilution for an extended period of time without any significant loss of melphalan. For example, a solid pharmaceutical composition comprising melphalan and a cyclodextrin derivative contains 2% or less, by weight, of a melphalan degradant after storage at 25° C. for a period of at least 2 years, or 5% or less, by weight, of a melphalan degradant after storage at 25° C. for a period of at least 3 years.

In some embodiments, a dry powder pharmaceutical composition of the present invention forms 2% or less of melphalan monohydroxide (based on a 100% initial concentration of melphalan) after storage for 2 years at room temperature.

Pharmaceutical Kits

The present invention is also directed to a pharmaceutical kit comprising a first container comprising 25 mg to 125 mg of melphalan as a hydrochloride salt and an optional water-soluble polymer, and a second container comprising an aqueous diluent, an optional buffer, and a cyclodextrin, derivative of formula I.

wherein n s 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are independently —H; a straight-chain or branched C₁-C₈-(alkylene)-SO₃ group, or an optionally substituted straight-chain or branched C₁-C₆ group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branched C₁-C₈-(alkylene)-SO₃ group; wherein, the cyclodextrin derivative is present in the, second container in a concentration of at least 50:1 (w/w) relative to the melphalan; and wherein combining the. first container and the second container provides a dilute pharmaceutical composition having a pH of about 4 to about 6 that degrades by 2% or less at about 25° C. within 5 hours after the diluting.

The present invention is also directed to a pharmaceutical kit comprising a first container comprising 150 mg to 250 mg of melphalan as a hydrochloride salt and an optional water-soluble polymer; and a second container comprising an aqueous diluent, an optional buffer, and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO3- group, or an optionally substituted straight-chain or branched C₁-C₆ group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight chain or branched C1-C8-(alkylene)-SO₃ group; wherein the cyclodextrin derivative is present in the second container in a concentration of 25:1 to. 35:1 (w/w) relative to the melphalan; and wherein combining the first container and the second container provides a dilute: pharmaceutical composition having a pH of about 4 to about 6 that degrades by 2% or less at about 25° C. within 5 hours after the diluting.

Alternatively, a first container comprises melphalan (in an amount described above) and a cyclodextrin derivative (as described above), an optional water-soluble polymer (e.g., povidone and the like), and an optional pH-adjusting agent; and a second container comprises a diluent (e.g., water, saline, and the like), an optional tonicity adjusting agent, and an optional pH-adjusting agent.

Materials suitable for use as the containers with the kits of the present invention include, but are not limited to, a glass (e.g., borosilicate glass, amber glass, and the like), a plastic (e.g., polypropylene, high-density polyethylene, poly(ethylene terephthalate, polystyrene, polycarbonate, and the like, and combinations thereof), a metal (e.g., a foil), and the like, and combinations thereof (e.g., a plastic-coated glass and/or metal).

Containers suitable for use with the pharmaceutical kits of the present invention include, but are not limited to, vials, bottles, sachets, and the like. The containers can be opened and/or the contents can be removed therefrom, by, for example, tearing, cutting, removing a screw-top, removing a stopper, piercing, squeezing, and the like, and combinations thereof.

In some embodiments, a first container comprises povidone in an amount of 10 mg to 30 mg, 15 mg to 25 mg, or about 20 mg. In some embodiments, a second container comprises a pH-adjusting agent (e.g., an acidifying agent, an alkalinizing agent, and/or a buffer) in a concentration sufficient to provide a pH of about 4 to about 6 when the first container and the second container are combined. In some embodiments, a second container comprises sodium citrate in an amount of 5.0 mg to 500 mg, 75 mg to 400 mg, 100 mg to 300 mg, 150 mg to 250 mg, or about 200 mg.

In some embodiments, the cyclodextrin derivative present in the second container is a compound of formula II:

wherein R═(H)_(21-x). or (—(CH₂)₄—SO₃Na⁺)_(x), and x=6.0-7.1; wherein the first container comprises about 200 mg of melphalan as a hydrochloride salt; and wherein the cyclodextrin derivative is present in the second container in an amount of about 27:1, about 30:1, or about 32:1 (w/w) relative to the melphalan.

Methods of Administering and Treating

In some embodiments, the present invention is directed to methods of delivering melphalan to a subject in need thereof, the method comprising administering a pharmaceutical composition or unit dosage form of the present invention to the subject in need thereof. The methods of the present invention include parenteral administration of the pharmaceutical compositions or unit dosage forms of the present invention.

In some embodiments, the pharmaceutical compositions or unit dosage forms: (or diluted forms thereof) are intravenously administered. Intravenous administration includes, but is not limited to, a. bolus injection, an intravenous infusion, a limb perfusion, a normothermic isolated limb infusion, a percutaneous hepatic perfusion, and the like, and combinations thereof. Administering the compositions of the present invention, can also be performed by injection and/or drip line using a cannula, a central line, a peripherally inserted central catheter line, and the like.

In some embodiments, a pharmaceutical composition of the present invention is administered as an infusion for a duration of 15 minutes to 6 hours, 30 minutes to 4 hours, 45 minutes to 3 hours, 1 hour to 2 hours, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 4 hours, about 5 hours, or about 6 hours.

In Some embodiments, the present invention is directed to parenterally administering a pharmaceutical composition or unit dosage form of the present invention to a subject for which an oral composition of melphalan is, for one or more reasons, not appropriate. For example, oral compositions of melphalan may not be appropriate because a subject may be too young, unable to swallow, undergoing surgery, incapacitated, or have a disorder that blocks absorption of melphalan administered via the oral route. Further, parenteral administration of the pharmaceutical compositions of the present invention are useful for treating conditions in subject in which a rapid increase in the in vivo concentration of melphalan is required.

In some embodiments, the present invention directed to methods of treating a subject suffering from a disease or disorder amenable to treatment with melphalan, the method comprising administering a pharmaceutical composition or unit dosage form to the subject. As used herein the terms “treat,” “treating,” and “treatment” refer to administering a composition of the present invention prior to the onset of clinical symptoms of a disease state/condition so as to prevent the development of any symptom, as well as administering the composition after the onset of one or more clinical symptoms of a disease state/condition so as to reduce or eliminate any such symptom, aspect or characteristic of the disease state/condition. Such treating need not be absolute to be useful. Additionally, the terms “treat” and “treatment” refer to both therapeutic treatment and prophylactic, maintenance, or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder or disease, or obtain beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of a symptom or a sign; diminishment of extent of a condition, disorder or disease; stabilization (i.e., not worsening) of the state of condition, disorder or disease; delay in onset or slowing of condition, disorder or disease progression; amelioration of a condition, disorder or disease state, remission (whether partial or total), whether detectable or undetectable; or enhancement or improvement of condition, disorder or disease. Treatment includes eliciting a clinically significant response, without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

As used herein, the term “subject” refers to warm blooded animals such as mammals, including humans and non-humans, such as, but not limited to, domestic and farm animals, zoo animals, sports animals, and pets (e.g., cats, dogs, mice, guinea pigs, horses, bovine cows, and sheep). In some embodiments, a subject is a human subject. Human subjects suitable for administering the pharmaceutical compositions and unit dosage forms of the present invention include, but are not limited to, pediatric, adult, and geriatric subjects. In some embodiments of the invention, the subject is a pediatric subject. For example, according to the U.S. Food and Drug Administration, a “pediatric” subject is up to 21 years of age, and includes neonates (birth to about 1 month of age), infants (about 1 month to about 2 years of age), children (about 2 to about 12 years of age) and adolescents (about 12 to about 21 years of age). See Guidance for industry and FDA Staff Premarket Assessment of Pediatric Medical Devices, U.S. Dept. of Health and Human Services, Food and Drug Administration, Center for Devices and Radiological Health, and Center for Biologics. Evaluation and Research (May 14, 2004). In some embodiments of the invention, the subject is an adult. As used herein, an “adult” subject is 18 years of age or older. In some embodiments, a subject is an adult that is about 50 years or older. In some embodiments of the invention, the subject is geriatric. Geriatric subjects are at least about 65 years of age. In some embodiments, a subject is about 70 years of age or older.

In some embodiments, the subject is a pediatric subject suffering from a disorder such as, but not limited to, an inborn defect, an immunodeficiency, a combined immunodeficiency, a severe combined immunodeficiency, a congenital neutropenia with defective stem cells, aplastic anemia, and combinations thereof.

In some embodiments, a subject is a geriatric subject scheduled to undergo a non-myeloablative procedure.

In some embodiments, the present invention comprises a method for treating a subject who has or is at risk for developing a condition amenable to treatment with melphalan, the method comprising administering an effective amount (i.e., a therapeutically effective amount) of a composition of the invention to the subject. Conditions amenable to treatment with melphalan include, but are not limited to, neoplastic disorders.

In some embodiments, a therapeutically effective amount for administering to a subject who has or is at risk for developing a condition amenable to treatment with melphalan is 25 mg to 125 mg, 40 mg to 110 mg, 40 mg to 75 mg, 40 mg to 60 mg, about 40 mg, about 50 mg, about 60 mg, about 75 mg, or about 100 mg of melphalan as a hydrochloride salt. The methods of the present invention also include titrating upward or downward from an initial melphalan dose in order to provide a therapeutically effective melphalan dosage. A therapeutically effective dose can be administered once, twice, thrice, four times, five times, six times, seven times, eight times, ten times, twelve times, or more as needed.

In some embodiments, the present invention is directed to a method of treating a disease, disorder or condition that is therapeutically responsive to a stem cell transplantation, the method comprising administering to a subject in need thereof a pharmaceutical composition or unit dosage form of the present invention followed by the subject undergoing a stern cell transplantation.

In some embodiments, a method of the present invention comprises administering a pharmaceutical composition or unit dosage form (or diluted form thereof) to a subject that suffers from a disorder selected from: myeloma, multiple myeloma, acute myelogenous leukemia, malignant melanoma, metastatic melanoma (e.g., metastatic ocular melanoma, metastatic cutaneous melanoma, and the like), breast cancer, ovarian cancer, testicular cancer, advanced prostate cancer, a myelodysplastic syndrome, a neuroendocrine cancer (e.g., a metastatic neuroendocrine tumor, and the like), a metastatic adenocarcinoma tumor, a hepatocellular carcinoma, osteogenic sarcoma, polycythemia veraplasma, plasma cell neoplasm, amyloidosis, scleromyxedema, and combinations thereof.

In some embodiments, a method of the present invention comprises administering a pharmaceutical composition or unit dosage form (or diluted form thereof) to a subject for whom a stem cell transplantation, has been indicated (e.g., a hematopoietic stem cell transplantation). In some embodiments, a subject for whom a stem cell transplantation has been indicated suffers from a disease or disorder selected from a leukemia, a cancer, a non-malignant disease, and combinations thereof. In some embodiments, a subject for whom a stem cell transplantation has been indicated suffers from a disease or disorder selected from myeloma, Multiple myeloma, a lymphoma, non-Hodgkin lymphoma (“NHL”), leukemia, acute myeloid leukemia (“AML”), Hodgkin's disease, acute lymphoblastic leukemia (“ALL”), a myelodysplastic syndrome (“MDS”), a myeloproliferative disorder (“MPD”), chronic myelogenous leukemia (“CML”), neuroblastoma, aplastic anemia, chronic granulocytic leukemia, a neuroblastoma, sickle-cell, disease, osteogenic sarcoma, Ewing's sarcoma, a desmoplastic small round cell tumor, plasma cell neoplasm, amyloldosis, scleromyxedema, and. combinations thereof. In some embodiments, a subject for whom a stem cell transplantation has been indicated is a subject who would not benefit from prolonged treatment with, or is already resistant to, chemotherapy.

Thus, the pharmaceutical compositions and unit. dosage forms of the present invention are useful for treatment of a condition amenable to treatment with melphalan, as well as use for conditioning a subject in need thereof for receiving a stem cell transplantation.

The amount of the pharmaceutical composition that is administered is therapeutically effective for the treatment that is desired. For example, a therapeutically effective amount for the treatment of multiple myeloma refers to an amount which, when administered, diminishes one or more symptoms associated with this disorder.

In some embodiments, the present invention is directed to a method of conditioning a subject in order to conduct a stem cell transplantation, the method comprising administering an effective amount of a pharmaceutical composition or unit dosage of the invention (e.g., intravenously) to the subject. Thus, the pharmaceutical compositions and unit dosage forms of the present invention are useful for treating a subject who suffers from a condition amenable to treatment by a stem cell transplant. As used herein, “stem cell transplantation” includes autologous and/or allogenic transplantation procedures.

The pharmaceutical compositions of the present invention are suitable for administering melphalan in a “high-intensity” or myeloablative conditioning regimen in preparation for a stem cell transplantation, or in a “reduced intensity” conditioning regimen in preparation for a stem cell transplantation. As used herein, “reduced intensity” conditioning refers to dosages in which a melphalan dose of less than 150 mg/m² is administered to a subject at any one dose. In some embodiments, the pharmaceutical composition of the present invention is administered to a subject that is 50 years of age or older who suffers from a condition amenable to treatment by a stem cell transplant.

In some embodiments, the melphalan is administered to a subject in need of a stem cell transplantation at a dose a 50 mg/m² to 300 mg/m², 50 mg/m² to 250 mg/m², 50 mg/m² to 225 mg/m², 50 mg/m² to 200 mg/m², 50 mg/m² to 175 mg/m², 50 mg/m² to 150 mg/m², 100 mg/m² to 300 mg/m², 100 mg/m² to 250 mg/m², 100 mg² to 225 mg/m², 100 mg/m² to 200 mg/m², 100 mg/m² to 175 mg/m², 100 mg/m² to 150 mg/m², 125 mg/m² to 300 mg/m², 125 mg/m² to 250 mg/m², 125 mg/m² to 225 mg/m², 1.25 mg/m² to 200 mg/m², 150 mg/m² to 300 mg/m², 150 mg/m² to 250 mg/m², 200 mg/m² to 300 mg/m², 200 mg/m² to 250 mg/m², about 50 mg/m², about 100 mg/m², about 125 mg/m², about 150 mg/m², about 175 mg/m², about 200 mg/m², about 250 mg/m², or about 300 mg/m².

In some embodiments, the administering comprises a dosage administered at four week intervals. In some embodiments, the dosage is administered twice, thrice, four times, five times, six times, eight times, or ten times. For example, in some embodiments a dose of about 100 mg/m² is administered three times with a four-week interval between the doses. In some embodiments, a dose of about 200 mg/m² is administered twice with a four-week interval between the doses. The final dose can be followed by a stem cell transplantation.

The pharmaceutical compositions and unit dosage forms of the present invention can be administered alone or in conjunction with, other medications or pharmaceutical compositions in some embodiments, a method of the present invention comprises to a subject a second therapeutic agent seeded from an alkylating agent other than melphalan, a platinum compound, an antimetabolite, a nitrosourea, a corticosteroid, a calcineurin inhibitor, a monoclonal antibody, a polyclonal antibody, a cytotoxic antibiotic, an interferon, an opioid, an antihistamine, a volume expander, a pressor agent, and combinations thereof. Additional second therapeutic agents include, but are not limited to, cisplatin, carboplatin, doxorubicin, bortezotmib, rituximab, thalidomide, gemeitabine, thiotepa, fludarabine, carmustine, etoposide, cytarabine, granulocyte colony-stimulating factor (G-CSF), ADH-1, topotecan, palifermin, prednisone, arsenic trioxide, ascorbic acid, busulfan, cyclophosphamide, N,N′,N″-triethylenethiophosphoramide, buthionine sulfoximine, and combinations, thereof. A second, therapeutic agent can be administered to a subject either in a pharmaceutical composition or unit dosage form, of the present invention that includes at least one additional therapeutic agent (in addition to melphalan), or as a separate pharmaceutical composition or unit dosage.

In some embodiments, the pharmaceutical compositions and/or unit dosage forms of the present invention are administered with other combinations of therapeutic active agents such as, but not limited to carmustine, etoposide, and cytarabine; busulfan and thiotepa; doxorubicin and bortezomib; arsenic trioxide and citric acid; thalidomide and rituximab; thalidomide and prednisone; and busulfan, fludarabine and G-CSF.

In seine embodiments, the pharmaceutical compositions and unit dosage forms of the present invention can enhance the bioavailability, rate of therapeutic onset, and/or therapeutic efficacy of melphalan. Thus, the present invention is also directed to a method of decreasing the time to therapeutic onset of melphalan following administration thereof, the method comprising orally or parenterally administering to a subject in need thereof a pharmaceutical composition or unit dosage form of the present invention. Wherein the time to therapeutic onset of melphalan provided by the orally or parenterally administered composition or unit dosages less than the time to therapeutic onset of melphalan provided by an orally administered reference composition that excludes the cyclodextrin derivative and contains an equivalent dose of melphalan. In some embodiments, the time to therapeutic onset of melphalan following administration of a pharmaceutical consumption or unit dosage form of the present invention is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, or at least 50% compared to the time to therapeutic onset of melphalan provided by an intravenously administered reference composition that excludes the cyclodextrin derivative and contains an equivalent, dose of melphalan.

In some embodiments, the dissolution of melphalan from the dosage forms of the present invention can be related to pharmacokinetic parameters and/or the in vivo concentration of melphalan and/or its metabolite(s). The in vivo concentration of melphalan and its metabolite(s), as well as pharmacokinetic parameters associated with an active form of melphalan can be determined by, e.g., sampling the blood plasma of a subject after administering a composition of the present invention. Pharmacokinetic parameters that can be measured include; but are not limited to, AUC_(0-t), AUC_(t-∞), AUC_(0-∞), and In(AUC_(LAST)).

As used herein, “AUC_(0-t)” refers to. the Area Under the Concentration time curve (i.e., plot of plasma concentration vs. time) after melphalan administration. The area is conveniently determined by the “trapezoidal rule”: the data points are connected by straight line segments, perpendiculars are erected from the abscissa to each data point, and the sum of the areas of the triangles and trapezoids so constructed is computed.

As used herein, “AUC_(t-∞)” refers to the Area Under the Concentration time curve, wherein the last concentration is extrapolated to baseline based on the rate constant for elimination.

As used herein, “AUC_(0-∞)” refers to the sum of the Area Under the. Concentration time curves for AUC_(0-t). and AUC_(t-∞).

As used herein, “In(AUC_(LAST))” refers to the Area Under the Concentration time curve determined by plotting plasma concentration on a natural logarithmic scale, using the last measured plasma concentration as the end point.

As used herein, “IntraCV” refers to an intra-assay coefficient of variation, which is the standard deviation within a sample set divided by the mean value of the sample set, with the result reported as a percentage.

In some embodiments, the bioavailability of melphalan in a human subject from a composition of the present invention is substantially greater than that observed upon administration of an equivalent dose of melphalan from a formulation lacking a cyclodextrin derivative (e.g., ALKERAN® for Injection ((GlaxoSmithKline) or Melphalan HCl Injectable (Bioniche Pharma USA)). For example, the dosage forms of the present invention can have an AUC_(0-t). or AUC_(0-∞), that is at least 20% or greater, at least 25% or greater, at least 30% or greater, at least 40% or greater, at least 50% or greater, at least 60% or greater, or at least 70% or greater than the AUC_(0-t) or AUC_(0-∞) observed after administration of a melphalan formulation to a subject that contains the same amount of melphalan and lacks a cyclodextrin derivative (e.g., ALKERAN® for Injection (GlaxoSmithKline) or Melphalan HCl Injectable (Bioniche Pharma USA)).

In some embodiments, the bioavailability of melphalan from a composition of the present invention is greater than that observed upon administration of an equivalent dose of melphalan from a formulation lacking a cyclodextrin derivative (e.g., ALKERAN® for Injection (GlaxoSmithKline) or Melphalan HCl Injectable (Bioniche Pharma USA)). For example, the dosage forms of the present invention can have an AUC_(0-t) or AUC_(0-∞) that is at least 20% or greater, at least 25% or greater, at least 30% or greater, at least 40% or greater, at least 50% or greater, at least 60% or greater, or at least 70% or greater than the AUC_(0-t) or AUC_(0-∞) observed after administration of a melphalan formulation to a subject that contains the same amount of melphalan and lacks a cyclodextrin derivative (e.g., ALKERAN® for Injection (GlaxoSmithKline) or Melphalan HCl Injectable (Bioniehe Pharma USA)). In some embodiments, the AUC_(0-t) or AUC_(0-∞) of melphalan from a composition of the present invention is 20% to 70%, 20% to 60%, 20% to 50%, 30% to 70%, 30% to 60%, 30% to 50%, 40% to 70%, 40% to 60%, or 50% to 70% greater than the AUC_(0-t) or AUC_(0-∞) observed upon administration of an equivalent dose of melphalan from a formulation lacking a cyclodextrin derivative (e.g ALKERAN® for Injection (GlaxoSmithKline) or Melphalan HCl Injectable (Bioniche Pharma USA)).

In some embodiments, the maximum plasma concentration (C_(max)) of melphalan from a composition of the present invention, is at least 20% or greater, at least 25% or greater, at least 30% or greater, at least 40% or greater, at least 50% or greater, at least 60% or greater, or at least 70% or greater than a C_(max) observed upon administration of an equivalent dose of melphalan from a formulation lacking a cyclodextrin derivative (e.g., ALKERAN® for Injection (GlaxoSmithKline) or Melphalan HCl Injectable (Bioniche Pharma USA)). In some embodiments, the maximum plasma concentration (C_(max)) of melphalan from a composition of the present invention is 20% to 70%, 20% to 60%, 20% to 50%, 30% to 70%, 30% to 60%, 30% to 50%, 40% to 70%, 40% to 60%, or 50% to 70% greater than C_(max) observed upon administration of an equivalent dose of melphalan from a formulation lacking a cyclodextrin derivative (e.g., ALKEIAN® for Injection (GlaxoSmithKline) or Melphalan HCl injectable (Bioniche Pharma. USA)).

In some embodiments, the rate of therapeutic onset of melphalan from a composition of the present invention is faster than that observed upon administration of an. equivalent dose of melphalan from a formulation lacking a cyclodextrin derivative (e.g., ALKERAN® for injection (GlaxoSmithKline) or Melphalan HCl Injectable (Bioniche Pharma USA)). For example, the dosage forms of the present invention have a time to C_(max) (i.e., t_(max)) that is. about 5%, about 10%, about 15%, about 20%, about 25%, or about 30% faster, or about 35% faster than a t_(max) observed upon administration of an equivalent dose of melphalan from a formulation lacking a cyclodextrin derivative (e.g., ALKERAN® for Injection (GlaxoSmithKline) or Melphalan HCl Injectable (Bioniche Pharma USA)). In some embodiments, the dosage forms of the present invention have a time to C_(max) (i.e., t_(max)) that is 5% to 35%, 5% to 30%, 5% to 25%, 5% to 20%, 10% to 35%, 15% to 35%, 20% to 35%, or 25% to 30% faster than a t_(max) observed upon administration of an equivalent dose of melphalan from a formulation lacking a cyclodextrin derivative (e.g., ALKERAN® for Injection (GlaxoSmithKline) or Melphalan HCl Injectable (Bioniche Pharma USA)).

In some embodiments, the pharmaceutical compositions of the present invention provide a reduced rate of hypersensitivity in patients after parenteral administration compared with patients parenterally administered, a similar dose of melphalan without a cyclodextrin derivative.

In some embodiments, the pharmaceutical compositions of the present invention provide a reduced rate of severe myelotoxicity in patients (e.g., patients who experience a white blood cell count <1,000 per mL and/or platelet count <25,000) after parenteral administration compared with patients parenterally administered a similar dose of melphalan without a cyclodextrin derivative.

In some embodiments, the pharmaceutical compositions of the present invention provide a reduced rate of death in, patients after parenteral administration compared with patients parenterally administered a similar dose of melphalan without a cyclodextrin derivative.

Raving generally described the invention; a further understanding can be obtained by reference to the examples provided herein. These examples are given for purposes of illustration, only and are not intended to be limiting.

EXAMPLES Example 1

The dissolution rate of free-base melphalan (Chemwerth, Woodbridge, Conn.) in solutions at various pH and at various concentrations of a cyclodextrin derivative were examined. The procedure was as follows: free base melphalan was added to a solution containing a cyclodextrin derivative (SBE_(6.5)-β-CD, CAPTISOL®) and then vortex mixed for 1-5 minutes, and, if necessary, sonicated in ice water until a clear solution was achieved.

TABLE Dissolution times for free base melphalan as a function of cyclodextrin derivative concentration, volume, and Target Melphalan Target Melphalan SBE_(6.5)-β-CD Dissolution Conc. Conc. Volume pH Tim (min) 50 mg/mL 200 mM 5 mL 5 50 50 mg/mL 125 mM 6 mL 5 90 50 mg/mL 100 mM 7 mL 5 160 50 mg/mL  75 mM 8 mL 5 >180 50 mg/mL  50 mM 10 mL  5 360 50 mg/mL 125 mM 6 mL 2.7 75 50 mg/mL 125 mM 10 mL  1.8 16 50 mg/mL 125 mM 6 mL 1.3 5 50 mg/mL 75, 100 125 mM 10 mL  1.1 <5 for all

Referring to the data in the above Table; the dissolution of free base melphalan was very rapid at pH 1.1 regardless of the concentration of the cyclodextrin derivative. After dissolution, the solution was then neutralized with sodium hydroxide.

The solutions of free base melphalan in this example can be prepared by addition of free base melphalan to a solution that contains the cyclodextrin derivative, or by adding a 0.1 M HCl solution to the melphalan and then adding the cyclodextrin derivative, or dissolving the free base melphalan and cyclodextrin derivative simultaneously. However, sonication was superior to mixing and/or shaking for dissolution enhancement and de-clumping of dry material in solution.

Example 2

The: binding of free-base: melphalan (Chemwerth, Woodbridge, Conn.) was studied as a function of cyclodextrin derivative concentration at pH 5 and pH 7, and the data was compared with literature reports of free-base melphalan binding. The pH solutions contained 100 mM sodium bitartrate buffer, and was adjusted to pH 5 using sodium hydroxide in 0.9% sodium chloride solution. The pH 7 solutions contained 50 mM each of mono- and di-basic phosphate and 0.9% sodium chloride. Solutions containing 0, 50, 75 and 100 mM SBE_(6.5)-β-CD (CAPTISOL®) were prepared, and excess free base melphalan was added to 2 mL samples of each solution. After the addition of free base melphalan, the samples were vortex mixed for 30 seconds, sonicated in ice water bath for 20 minutes, and then mixed by end-over-end rotation at room temperature for 30 minutes. The samples were then centrifuged, the clear supernatant was diluted with water, and analyzed by HPLC.

All melphalan assays performed by HPLC utilized the following protocol. A Shimadzu HPLC equipped with a SCL-10A system controller, SIL-10A auto injector, LC-10AT liquid chromatograph, SPD-10A UV spectrophotometer detector, CTO-10A column oven, and Class-VP chromatography laboratory automated software was utilized. The column was a ZORBAX® RX-C18 4.6 mm by 150 mm column (Agilent Technologies) having a 5 μm particle size. Samples were injected (20 μL) onto the column for isocratic elution using mobile phase of phosphate buffered saline (pH 7.4):methanol:glacial acetic acid in a ratio of 500:250:10 (v/v). The mobile phase was selected in order to decrease or nominally quench melphalan conversion by having a high-chloride concentration. The samples were prepared immediately prior to injection. Detection was at 260 nm.

The literature procedure involved adding an excess amount of melphalan to 0, 10, 20, 30, 40, 50, 75 and 100 mM solutions of SBE₇-β-CD (avg. M.W.=2248 g/mol) in a 25 mM phosphate buffer solution at pH 7.5. The suspensions were placed in tightly capped vials, sonicated 1 h, and agitated at 25° C. for 23 h. The solutions were then centrifuged, the clear supernatant was diluted with doubly distilled water, and analyzed by HPLC. See D. Q. Ma et al., J. Pharm. Sci. 89:275 (1999);

The data from the free base melphalan solubilization studies are provided in FIG. 1. Referring to FIG. 1, free base melphalan displayed a significantly lower solubility enhancement than that provided in a previous literature report. See id. Because the solubility enhancement of free base melphalan provided by SBE_(6.5)-β-CD was lower than expected, additional phase solubility tests were performed using melphalan hydrochloride.

Example 3

The binding of melphalan hydrochloride (USP reference standard) and free base melphalan (Chemwerth) with a cyclodextrin derivative (SBE_(6.5)-β-CD, CAPTISOL®, avg. M.W.=2163 g/mol) was determined as a function of cyclodextrin derivative concentration at pH 7.5. The temperature was maintained at 22° C., and a 25 mM phosphate buffer was added to each solution. The data was compared with a literature report of free-base melphalan binding with SBE₇-β-CD (avg., M.W.=2248 g/mol) in a 25 mM phosphate buffer at pH 7.5 (see Example 2).

The samples were prepared by adding excess melphalan hydrochloride, or free base melphalan to a 1 mL sample of various SBE_(6.5)-β-CD solutions. The samples were vortex mixed for 30 seconds, sonicated at 20-24° C. for 60 minutes, and then mixed by end-over-end rotation at 22° C. for 60 minutes. The samples were then centrifuged, the clear supernatant was diluted with water, and analyzed by HPLC. The data are provided in FIG. 2. Referring to FIG. 2, the melphalan hydrochloride salt displayed a significant solubility enhancement compared to free base melphalan for all cyclodextrin derivative concentrations above 25 mM.

Example 4

A pharmaceutical composition comprising melphalan as a hydrochloride salt was prepared by the process outlined schematically in FIG. 3. Referring to FIG. 3, water for injection, USP was placed in a stainless steel, mixer at a temperature of 15-20° C., and hydrochloric acid was added until a pH of about 4.6 was achieved. The resulting solution was stirred at a speed sufficient to produce a vortex (but without foaming or frothing) for about 15 minutes, a cyclodextrin derivative (27.2 g SBE_(6.5)-β-CD, CAPTISOL®) was added slowly while vortex stirring, and the resulting solution was stirred for about 15 minutes to ensure complete dissolution. The resulting solution had a pH of about 2.5. Melphalan as hydrochloride salt (516 mg) was added slowly while vortex stirring, and the resulting solution was stirred for about 15 minutes to ensure complete dissolution. A base (2 N NaOH) was then slowly added while vortex stirring until the solution had a pH of about 5.6. The solution, was then assayed using a UV/vis spectrophotometer (detection wavelength of 260 nm). The solution comprised melphalan at a concentration of 5.16 mg/ML, and the melphalan was present in a ratio of about 1:55 w/w relative to the cyclodextrin derivative. The solution was then passed through a sterile filter (0.22 μm PVDF) and cooled to 10-15° C.

Example 5

The liquid pharmaceutical composition provided in Example 4 was lyophilized to provide a reconstitutable and/or dilutable dry powder comprising 50 mg of melphalan as a hydrochloride salt. Glass Vials were filled with the solution (10 mL) and placed in trays on a pre-cooled shelf at 5° C. The vials were allowed to thermally equilibrate for about 30 minutes, and were then lyophilized to provide a dry powder in each vial. The vials were back-filled with nitrogen at a pressure of about 400 mTorr, and then sealed.

Example 6

A pharmaceutical composition comprising melphalan as a hydrochloride salt was prepared by the process described in Example 4, and outlined schematically in FIG. 3, except that the final solution contained melphalan at a concentration of 10 mg/mL, and the melphalan was present in a ratio of about 1:27 w/w relative to the cyclodextrin derivative.

Example 7

The liquid pharmaceutical composition provided in Example 6 was lyophilized to provide a reconstitutable and/or dilatable dry powder comprising 200 mg of melphalan as a hydrochloride salt. Glass vials were filled with the solution (20 mL) and placed in trays on a pre-cooled Shelf at 5° C. The vials were allowed to thermally equilibrate for about 30 minutes, and were then lyophilized to provide a dry powder in each vial. The vials were back-filled with nitrogen at a pressure of about 400 mTorr, and then sealed, packaged, and labeled. The vials were protected from exposure to light during all aspects of the lyophilization, back-filling, sealing, packaging and labeling procedures.

Prophetic Example A

The liquid pharmaceutical composition provided in Examples 4 and 6 will be aseptically spray dried to provide a free-flowing powder to be filled aseptically. The free-flowing powder will meet or exceed the dissolution properties of the lyophilized powder prepared in Examples 4 or 6.

Example 8

A pharmaceutical composition comprising melphalan as a hydrochloride salt was prepared by the process outlined schematically in FIG. 4. Referring to FIG. 4, water for injection, USP was placed in a stainless steel mixer at a temperature of 18-25° C., and the resulting solution was stirred at a speed sufficient to: produce a vortex (but without foaming or frothing). A cyclodextrin derivative (SBE_(6.5)-β-CD, CAPTISOL®) was added slowly while vortex stirring, and the resulting solution was stirred for about 15 minutes to ensure complete dissolution. The resulting solution was then cooled to about 2-8° C. Melphalan as a hydrochloride salt was added slowly while vortex stirring, and the resulting solution was stirred for about 15 minutes to ensure complete dissolution. A base (2 N NaOH) was then slowly added while vertex stirring until the solution had a pH of about 5-6 (target pH 5.5). An in-process control (“IPC”) assay was then, performed to monitor pH, and the solution was diluted to the final target volume using water for injection, USP. The solution was then assayed using a UV/vis spectrophotometer (detection wavelength, of 260 nm) and a bioburden assay was performed. The solution was then passed through a sterile filter (0.22 μm PVDF) and cooled. to 15-25° C. Finally, an IPC assay was conducted.

Example 9

The solution prepared in Example 8 was lyophilized to provide a reconstitutable and/or dilutable dry powder comprising melphalan as a hydrochloride salt. For the lyophilization, glass vials were filled with the solution (10 mL) and placed in trays on a pre-cooled shelf at. 5° C. The vials were allowed to thermally equilibrate for about 1 hour, and were lyophilized to provide a dry powder in each vial. The vials were back-filled with nitrogen, sealed, packaged, and labeled. The vials were protected from exposure to light during all aspects of the lyophilization, back-filling, sealing, packaging and labeling procedures.

Prophetic Example B

The liquid pharmaceutical composition provided in Example 8 will be aseptically spray dried to provide a free-flowing powder to be filled aseptically. The free-flowing powder will meet or exceed the dissolution properties of the lyophilized powder prepared in Example 9.

Example 10

The properties of the pharmaceutical compositions of the present invention after dilution with Water for Injection, USP, were analyzed by a variety of analytical methods. The results are listed in the Table below. Compositions A-D were prepared by the process described in Examples 8-9. The diluted compositions contained SBE_(6.5)-β-CD (CAPTISOL®) in a concentration of 75 mM, 100 mM, 125 mM, and 125 mM, respectively. Each of the compositions had a moisture content of about 1.3% to about 2.5% prior to dilution.

TABLE Properties of pharmaceutical compositions of the present invention containing varying concentrations of a cyclodextrin derivative. Dilution SBE_(6.5)-β-CD Dissolution Density Time for Volume Conc. Time pH (22° C.) Viscosity 5% loss A 10 mL  75 mM <30 s 5.05 1.07 g/cc 2.06 cP 10 h B 10 mL 100 mM <30 s 4.9 1.08 g/cc 2.28 cP 23 h C 10 mL 125 mM  45 s 5.05 1.11 g/cc 2.95 cP 49 h D  5 mL 125 mM 105 s 5.2 1.11 g/cc 3.02 cP 25 h

Example 11

The stability of melphalan hydrochloride upon dilution of a. pharmaceutical composition of the present invention was determined. Pharmaceutical compositions containing a cyclodextrin derivative (SBE_(6.5)-β-CD, CAPTISOL®, avg. M.W.=2163 g/mol) were diluted with isotonic saline to provide 0.45 mg/mL melphalan solutions that contained the cyclodextrin derivative at a concentration of 75 mM and 125 mM, respectively. Melphalan was assayed as a function of time, and the time necessary for a 5% or 10% loss of melphalan (based on an initial melphalan concentration of 100%) was determined. The data is provided in the Table below. The times required for melphalan to fall to 90% or 95% of its initial concentration in the solutions that contained the cyclodextrin derivative were compared to the stability of melphalan in a Reference product (ALKERANR for Injection, GlaxoSmithKline).

TABLE Melphalan stability as a function of cyclodextrin derivative concentration, compared to a reference melphalan standard. Time for 5% loss Time for 10% loss SBE6.5-β-CD (75 mM) 5.4 h 11 h SBE6.5-β-CD (125 mM) 8.8 h 18 h Reference 1.3 h 2.7 h 

Referring to the data in the above Table, the stability of melphalan after dilution from a pharmaceutical composition of the present invention shows an improvement of approximately 4.2 times and 6.8 times at a cyclodextrin derivative concentration of 75 mM and 125 mM, respectively, compared to a reference melphalan formulation that does not contain a cyclodextrin derivative.

Example 12

The stability of melphalan hydrochloride upon dilution of a pharmaceutical composition of the present invention was determined as a function of temperature and storage conditions. Pharmaceutical compositions containing melphalan (50 mg) and a cyclodextrin derivative (SBE_(6.5)-β-CD, CAPTISOL®, avg. M.W.=2163 g/mol, 270 mg) were diluted with isotonic saline (8.5 mL) to provide a concentrated solution. The concentrated solution was further diluted 10-fold to provided a dilute solution. Each of the concentrated and diluted melphalan solutions were stored at 25° C./60. relative humidity, or in a refrigerator (˜10° C.), and the melphalan content was monitored as a function of time. The data is provided in the Table below.

TABLE Melphalan stability as a function of temperature and storage conditions. Storage Melphalan Monohydroxy Solution Time Conditions Assay Melphalan Assay Conc. Sol'n 0 Refrigerator 99% 0.8% ″ 6.5 ″ 98.9%  0.8% ″ 24.5 ″ 98.8%  0.9% ″ 48.5 ″ 98.4%   1% Conc. Sol'n 0 25° C./60% R.H. 99% 0.8% ″ 6 ″ 98% 1.5% ″ 24 ″ 96% 3.4% ″ 48 ″ 93% 5.7% Dilute Sol'n 0 Refirgerator 99%  1% ″ 5 ″ 98.4%  1.4% ″ 24.3 ″ 97.8%   2% ″ 48.4 ″ 96.7%  2.7% Dilute Sol'n 0 25° C./60% R.H. 99%  1% ″ 5.3 ″ 94% 5.3% ″ 23.8 ″ 81% 15.5%  ″ 47.2 ″ 70% 20.2% 

Referring to the data in the above Table, the stability of melphalan after dilution from a pharmaceutical composition of the present invention provides a significant improvement compared to currently available melphalan pharmaceutical compositions that do not contain a cyclodextrin derivative.

Example 13

The stability of a lyophilized melphalan hydrochloride composition was determined before and after dilution as a function. of temperature and storage conditions. The study was performed under the direction of CyDex Pharmaceuticals, Inc. by BioConvergence LLC, Bloomington,

Compositions comprising melphalan (50 mg) and povidone (20 mg) were diluted with compositions comprising a cyclodextrin derivative (SBE_(6.5)-β-CD, CAPTISOL®, avg. M.W.=2163 g/mol, 270 mg), sodium, citrate (200 mg), and distilled water (10 mL) to provide concentrated melphalan solutions (5 mg/mL). In addition to testing the stability of the concentrated solutions, further dilution 11-fold to provide dilute solutions containing melphalan (0.45 mg/mL).

The kinetic stability of a reconstituted concentrated solution (5 mg/mL melphalan) was determined upon storage in a glass vial, and the kinetic stability of a reconstituted diluted solution (0.45 mg/mL melphalan) was determined upon storage in a 50 mL Baxter INTRAVIA® bag, at refrigerated (about 2°-8° C.) and room (about 25° C., monitored under fluorescent light) temperatures.

Evaluation of the kinetic stability of the dilute (0.45 mg/ml melphalan) composition was determined in a 50 mL Type I glass container: reconstitution was performed using saline (8.5 mL), and aliquots (4.5 mL) were removed from each vial and injected into 4 glass containers that held 45.5 mL of saline. After an amount was withdrawn from each container for t=0 analysis, the containers were stored at room temperature (“RT”, about 25° C., under fluorescent light), or in a refrigerator (about 2°-8° C.), and the melphalan content was monitored as a function of time. The data is provided in the Table below.

TABLE Summary of melphalan stability as a function of temperature and storage conditions MEL Hold Degradation Run Form Storage Conditions Time (% w/w) 1 Lyophilized comp. RT (~20°-25° C.) 2 yrs <2% Conc. Solution (5 mg/mL) Immediately diluted n/a n/a Dilute solution (0.45 mg/mL) RT (~20°-25° C.) 10 h <4% 2 Lyophilized comp. RT (~20°-25° C.) 2 yrs <2% Conc. Solution (5 mg/mL) RT (~20°-25° C.) 24 h <4% Dilute solution (0.45 mg/mL) RT (~20°-25° C.) 5 h <2% 3 Lyophilized comp. RT (~20°-25° C.) 2 yrs <2% Conc. Solution (5 mg/mL) Refrigerated (2°-8° C.) 48 h <4% Dilute solution (0.45 mg/mL) RT (~20°-25° C.) 5 h <2% 4 Lyophilized comp. RT (~20°-25° C.) 2 yrs <2% Conc. Solution (5 mg/mL) immediately diluted n/a n/a Dilute solution (0.45 mg/mL) 1) Refrigerated (2°-8° C.) 1) 24 h 1) <2%   2) RT (~20°-25° C.) 2) 5 h 2) <2%  

Referring to the data in the above Table, the stability of melphalan after dilution from a pharmaceutical composition of the present invention provides a stable composition that can be maintained at room temperature for up to 5 hours and exhibit less than 2% melphalan degradation, or up to 10 hours and exhibit less than 4% melphalan degradation. When refrigerated, a diluted melphalan composition can be stored up to 24 hours and exhibit less than 2% melphalan degradation. Additionally, a lyophilized pharmaceutical composition can be stored up to 2 years at room temperature and exhibit less than 2% melphalan degradation.

Furthermore, referring to Examples 12 and 13, the pharmaceutical compositions of the present invention provide a significant improvement in melphalan stability compared to other formulations in which the use of a cyclodextrin derivative has been proposed. For example, D. Q. Ma et al., Int. J. Pharm. 189:227 (1999) provide a melphalan composition that upon dilution with a solution containing a cyclodextrin derivative, exhibits a melphalan loss of more than 60% after 48 hours at room temperature. Significantly, the data in the above Tables illustrates that upon dilution of a pharmaceutical composition of the present invention, a melphalan loss of at most 30% is observed within 45 hours at room temperature. The melphalan loss can be reduced to as low as 2% or 3% when the solution is stored at a reduced temperature (e.g., in a refrigerator).

Example 14

The hemolytic potential of a cyclodextrin derivative suitable for use with the pharmaceutical composition of the present invention was analyzed in comparison to a previously marketed diluent vehicle for melphalan (ALKERAN® For injection, GlaxoSmithKline). The hemolytic potential was evaluated in rodent (SPRAGUE DAWLEY® or Wistar Han IGS rats) and human red blood cells obtained from fasted subjects using a spectrophotometric technique. Normal saline (0.9% sodium chloride) was used as the blank (or background) and as a negative control for comparison against various concentrations of a cyclodextrin derivative-containing, and cyclodextrin-free diluent vehicles. A positive control containing Triton X-100 (1%) in phosphate buffered saline was also utilized. Human red blood cells were taken from fasted (≥8 h) adult subjects. The components of the various samples are listed in the following

TABLE Components of diluent vehicles used for hemolysis studies. Identification Constituents Negative Control/Bank 0.9% Sodium Chloride Positive Control 1% TRITON ® X-100 in phosphate buffered saline Cyclodextrin Derivative SBE_(6.5)-β-CD (CAPTISOL ®, 9.72 g) Diluent q.s. 400 mL with normal saline ALKERAN ® for Injection Povidone (K-12, 72 mg) (GlaxoSmithKline) Diluent Sodium citrate (720 mg) Propylene glycol (21.6 mL, 22.4 g) Ethanol (1.87 mL, 1.48 g) Water (12.2 mL) q.s. 400 mL with normal saline

The rat and human red blood cells were exposed to various concentrations of the diluent vehicles and the hemolytic potential was evaluated using. equation (1):

A ₅₄₁(test article)−A ₅₄₀(negative control)×100=% Hemolysis  (1)

The hemolysis results are provided in the following Table, where Group A refers to rat red blood dells exposed to the cyclodextrin derivative vehicle; Group B refers to rat red blood cells exposed to the ALKERAN® for injection (GlaxoSmithKline) diluent vehicle; Group C refers to human red blood cells exposed to the cyclodextrin derivative vehicle; and Group D refers to human red blood cells exposed to the ALKERAN® for Injection (GlaxoSmithKline) diluent vehicle. The negative controls for each experiment provided absorbances below 0.13, and the positive controls for each experiment provided absorbances of about 2.8 to 3.

TABLE Hemolysis results for rat and human red blood cells exposed to various diluent vehicles. Dilution Group Meas. none 1:2 1:4 1:8 1:16 1:32 1:64 1:128 A Abs. (a.u.) 0.11 0.114 0.11 0.117 0.116 0.118 0.119 0.118 Hem. (%) 1% 0 0 0 0 0 0 0 B Abs. (a.u.) 0.178 0.155 0.141 0.128 0.125 0.121 0.123 0.124 Hem. (%) 2% 1% 1% 0 0 0 0 0 C Abs. (a.u.) 0.021 0.021 0.022 0.019 0.017 0.016 0.017 0.025 Hem. (%) 0 0 0 0 0 0 0 0 D Abs. (a.u.) 0.037 0.031 0.033 0.02 0.025 0.022 0.027 0.027 Hem. (%) 0 0 0 0 0 0 0 0

Referring to the hemolysis data in the above Table, the Solution that contained the cyclodextrin: derivative at high concentrations (e.g., no dilution, 1:2 dilution′ with saline, and 1:4 dilution with saline) provided reduced hemolysis in rat red blood cells, which was also exhibited as reduction in spectrophotometric absorption of about 30% compared to the ALKERAN® for Injection (GlaxoSmithKline) diluent. While, the hemolysis tests in human blood cells exhibited, a similar reduction in spectiophototnetrie absorbance at high concentrations, neither the cyclodextrin derivative solution or the ALKERAN® for Injection (GlaxoSmithKline) diluent vehicle induced homolyses in human red blood, cells.

Example 15

A study was conducted that determined melphalan associated with SAE_(6.5)-β-CD (CAPTISOL®, CyDex Pharmaceuticals, Inc., Lenexa, Kans.) exhibits the same protein binding as unassociated melphalan. The study was performed under the direction of CyDex Pharmaceuticals, Inc. by Analytical Biochemistry Laboratories, Inc., Columbia, Mo.

Preliminary Study

A preliminary study was performed that determined radioactive-labeled melphalan, [¹⁴C]-melphalan (Moravek Biochemicals, Inc., Brea, Calif.), does not bind non-specifically to ultrafiltration devices. The following mixtures of compounds were added to human plasma ultrafiltrate (Biochemed, Winchester, Va.) to determine the protein binding of [¹⁴C]-melphalan alone or in combination with SAE_(6.5)-β-CD:

-   -   1. [¹⁴C]-melphalan with melphalan; and     -   2. [¹⁴C]-melphalan with melphalan and SAE_(6.5)-β-CD.

Radioactive-labeled warfarin, [³H]-warfarin (Moravek Biochemicals, Inc., Brea, Calif.), a compound with well-documented protein binding properties was used as a positive control in all experiments.

Powdered material (as applicable) was weighed into scintillation vials (20 mL) and the radiolabeled compounds were added to the vials using a positive displacement pipette. Blank human plasma ultrafiltrate (5 mL) was then added to the vials using a glass serological pipette. The mixtures were then blended briefly. The time-dependence of plasma protein binding was determined by sampling the mixtures 0.5, 1, and 5 minutes after addition of the test compounds to the human plasma ultrafiltrate. The sample aliquots (3×1 mL) were dispensed into the ultrafiltration devices, and the samples were immediately centrifuged (1600 g for 5 minutes at 25° C.). The solution remaining in the vials was then aliquoted for Liquid Scintillation Counting (LSC) analysis (2×0.1 mL).

More than 95% recovery was observed for [³H]-warfarin in all experiments. In the protein binding experiments, [3H]-warfarin was over 99% protein bound. Radiolabeled melphalan (alone or in combination with. SAE_(6.5)-β-CD) applied to ultrafiltration devices having a molecular weight cutoff of 30 kD exhibited an average of over 97% radioactivity recovery: [¹⁴C]-melphalan alone exhibited a recovery of 97.7% (n=3), and [¹⁴C]-melphalan with SAE_(6.5)-β-CD exhibited a recovery of 97.6% (n=3). The results demonstrate that there was minimal (i.e., less. than 2.4%) non-specific binding of radiolabeled melphalan to the ultrafiltration devices.

Protein-Binding Study

For the protein-binding study, the radiolabeled melphalan, non-radiolabeled melphalan, and SAE_(6.5)-β-CD (as applicable) were added to a scintillation vial (20 mL) and blown to dryness under a nitrogen stream to standardize the amount of solvent present in each experiment. Methanol (50 μL) was added to the vials and blank human plasma (5 mL) was added to the vials using a glass serological pipette. The mixtures were then blended briefly. Aliquots (3×1 mL) were then dispensed from the vials into the ultrafiltration devices, followed by centrifugation (2,000 g for 5 minutes at 25° C.). The time interval between adding human plasma to the vials and the start of centrifugation was 0.5, 1, 5, 10, and 30 minutes. The solution remaining, in the vials was then aliquoted in duplicate (at a volume of 0.1, 0.05, and 0.025 mL) for LSC analysis.

Sample radioactivity was quantified using a scintillation counter (Beckman Instruments, Inc. Schaumberg, Ill.) equipped with the H-number method for cpm to dpm conversion. LSC analysis was performed with samples (5 mL) in glass scintillation vials (7 mL), from which background measurements were made using the same amount of scintillation fluid added to the vials. The results are provided in the following Table:

TABLE Protein binding of radiolabeled melphalan (“[¹⁴C]-mel”) with unlabeled melphalan (“mel”) in the presence and absence of SAE_(6.5)-β-CD. Total Conc. Conc. Recovery (%) % Binding Mixture (dpm/mL) (μg/mL) Rep. Data Avg. Data Avg. [¹⁴C]-mel + mel 112,105 14 1 96.4 97.1 64.5 64.3 ± 0.34 (1 min.) 2 96.9 63.9 3 98.0 64.4 [¹⁴C]-mel + mel + 112,382 14 1 92.9 91.5 63.7 64.3 ± 0.69 SAE_(6.5)-β-CD 2 95.4 64.1 (0.5 min.) 3 86.4 65.1 [¹⁴C]-mel + mel + 108,085 13 1 97.3 97.5 63.8 64.4 ± 0.59 SAE_(6.5)-β-CD 2 97.0 64.8 (1 min.) 3 98.3 64.6 [¹⁴C]-mel + mel + 109,621 13 1 96.5 95.9 65.8 67.2 ± 1.96 SAE_(6.5)-β-CD 2 95.7 69.5 (5 min.) 3 96.5 66.4 [³H]-warfarin 114,932 0.052 1 98.5 101 98.9 98.8 ± 0.01 2 100 98.8 3 103 98.8

The results showed that [¹⁴C]-melphalan in the absence of SAE_(6.5)-β-CD was 64.3% protein bound after 1 minute in human plasma. Similar degrees of protein binding were observed for [¹⁴C]-melphalan in the presence of SAE_(6.5)-β-CD, 64.3% (0.5 minutes, n=3), 64.4% (1 minute, n=3), and 67.2% (5 minutes, n=3). The study showed that SAE_(6.5)-β-CD did not affect the protein-binding of radiolabeled [¹⁴C] melphalan.

Example 16

A study was performed to investigate the potential for a sulfoalkyl either cyclodextrin to perturb the in vivo pharmacokinetics of melphalan. Pharmacokinetic parameters were determined for melphalan following intravenous administration to male Sprague Dawley rats in the presence or absence of SBE_(6.5)-β-CD in the delivery vehicle.

The pharmacokinetics of melphalan were studied in overnight-fasted male Sprague Dawley rats. All experimental procedures were approved, and performed in accordance with the guidelines of the Institutional Animal Experimentation Ethics Committee (Monash University Ethics approval number VCPA/2008/02).

On the day prior to dosing, a commercially available BASi CULEX® cannula (for use with a CULEX® automated blood sampling device) was inserted into the left carotid artery of each rat under isoflurane anesthesia (2%). A polyethylene cannula was also inserted into the right jugular vein. Cannulae were exteriorized by tunneling subcutaneously to emerge above the scapulae.

Immediately following surgery and through to the end of the experiment, rats were housed in RATURN® metabolic cages in the CULEX® automated blood sampler. All rats returned to normal grooming, drinking and sleeping behavior within an hour of surgery. Animals were given a small amount of food just after they awoke from the anesthetic, but were then fasted for 16-18 hours prior to drug administration. Animals had access to water ad libitum at all times. Food was reinstated 4 hours following drug administration. At the conclusion of each experiment, rats were killed by a single lethal injection of pentobarbitone.

The cyclodextrin derivative-free melphalan formulation was prepared as per the product insert for ALKERAN® for Injection (GlaxoSmithKline). The contents of a single ALKERAN® for Injection (GlaxoSmithKline) vial were reconstituted with 10 mL of sterile diluent (provided with the ALKERAN® product, and containing 0.2 g sodium citrate, 6 mL propylene glycol, 0.52 mL ethanol (96%) and water). The solution was then further diluted with 0.9% normal saline (2 mL ALKERAN® for Injection (GlaxoSmithKline) in 10 mL of 0.9% saline, i.e., 12 mL total volume), and the resultant formulation was sterilized by filtering through a 0.22 μm syringe filter before administering to rats. The measured concentration of melphalan in the IV formulation was 0.54 mg/mL (as free base) and the pH of the final solution was between 5 and 6 (checked using pH paper). The formulation was administered to animals within 30 minutes of preparation.

A formulation containing SBE_(6.5)-β-CD (27% w/v) was prepared by dissolving SBE_(6.5)-β-CD in Milli-Q water. Contents of an ALKERAN® for Injection (GlaxoSmithKline) vial were then reconstituted with 10 mL of the SBE_(6.5)-β-CD solution. This solution was then diluted with 0.9% saline (2 mL in 10 mL of 0.9% saline) and the resultant formulation was sterilized by filtering through a 0.22 μm syringe filter before administering to rats. Thus, the final formulation contained. 4.5% (w/v) SBE_(6.5)-β-CD. The measured concentration of melphalan (as free base) was 0.58 mg/ml, and the pH of the final solution was between 5 and 6 (checked using pH paper). The formulation was administered to animals within 30 minutes of preparation.

The total dose volume was 1 mL and all doses were infused manually via the jugular vein cannula. The complete dose was delivered over a 10-minute period and the cannula was flushed with heparinized saline (10 U/mL) to ensure complete administration of the dose.

Samples of arterial blood and urine were collected according: to the following schedules: blood/plasma sampling times were pre-dose, and 5, 10 (end of infusion), 15, 25, 40, 55, 70, 100, 130, 190, 250, 370, and 490 minutes post-dose; and urine sampling intervals were 0-70 minutes, 70-130 minutes, 130-190 minutes, 190-250 minutes, 250-310 minutes, 310-370 minutes, 370-430 minutes, 430-490 minutes, and 490-1450 minutes.

Arterial blood was collected directly into borosilicate vials (at 4° C.) containing heparin, COMPLETE® (a protease inhibitor cocktail), potassium fluoride, and EDTA to minimize potential for ex vivo degradation in blood/plasma samples. Once collected, an aliquot (50 μL) of whole blood was transferred into a fresh micro-centrifuge tube. The remaining blood was centrifuged and supernatant plasma removed. All blood, plasma and urine samples were immediately (snap) frozen on dry ice and then transferred to −20° C. freezer for storage until analysis.

The melphalan concentration in whole blood, plasma, urine and samples of dosing solutions were determined using LC-MS.

Sample preparation was performed using protein precipitation with acetonitrile. Aliquots of the plasma and blood (50 μL) were treated with internal standard (10 μL), acetonitrile (130 μL), vortexed and centrifuged. The supernatant was removed and analyzed by LC/MS. Standard samples were prepared by spiking solution standards in the respective blank matrix. A stock solution of melphalan free base was prepared at a concentration of 10 mg/mL in dimethylsulfoxide. This stock solution was further diluted in aqueous acetonitrile (50% v/v) to obtain spiking solutions for the preparation of calibration standards.

All samples were assayed via LC-MS/MS on a Micromass Quattro Premier PR triple quadrupole instrument coupled with a Waters Acquity UPLC (Waters Corp., Milford, Mass.). Analytical separations were performed on a Phenomenex Polar reverse-phase column (50 mm^(x)1.0 mm inner diameter, 4 μm particle size) equipped with a Phenomenex Polar Security Guard column of the same material (Torrance, Calif.). Samples (7.5 μL) were injected onto the column and compounds were eluted (at a flow rate of 0.15 mL/min) using a ternary gradient solvent system consisting of an aqueous solution of methanol (2% v/v) and formic acid (0.05% v/v) Milli-Q water (solvent A), and acetonitrile containing formic acid (0.05% v/v) (solvent B). The gradient conditions used for LC-MS analysis are listed in the following table.

TABLE Gradient chromatography conditions used for the analysis of melphalan. Time (minutes) Solvent A Solvent B  0-0.2 89 2 0.3 80 20 2.7 20 80 2.8 5 95 3.3 5 95 3.5-4.5 98 2

Elution of analytes was confirmed by multiple reaction monitoring (MRM) using diazepam (0.2 μg/mL) as the internal standard (diazepam). Inlet cone voltages were 20 eV and 40 eV for melphalan and the internal standard, respectively, and collision energies of 15 eV and 27 eV for melphalan and the internal standard, respectively. The elution of melphalan and internal standard was monitored using the following transitions. 304,94>26788 and 285.17>154.02, respectively. Melphalan exhibited a retention time of 2.0 minutes and the internal standard exhibited a retention time of 2.8 minutes,

Mass spectrometry was performed using positive mode electrospray ionization with a capillary voltage a 3.2 kV, detector Multiplier gain of 650 V, and source block and desolvation temperatures of 90° C. and 300° C., respectively. A desolvation gas (nitrogen) and collision gas (argon) flow of 500 L/h and 0.38. mL/min, respectively, was maintained. The lower limit or quantitation (LLQ) for blood and plasma standards was 5.0 ng/mL, and the LLQ for diluted urine samples was 0.5 ng/mL.

Both plasma and blood concentration data were analysed to obtain pharmacokinetic parameters using WINNONLIN® software WINNONLIN® professional version 5,2,1, Pharsight Corp., Mountain View, Calif.). The total clearance (CL_(total), for whole blood or plasma) after intravenous administration was calculated as: CL_(total)=Dose/AUC, where AUC is the area under the whole blood or plasma concentration versus time curve obtained using the linear trapezoidal method. The volume of distribution (V_(z)) was calculated as V_(z)=/λ_(z), where CL_(total)/λ_(z), where λ_(z) is the elimination rate constant after i.v. administration.

The mean dose-normalized concentration versus time profiles of melphalan in whole blood and plasma following intravenous administration using a formulation containing SBE_(6.5)-β-CD (n=4) and a cyclodextrin-free (n=5) formulation are presented in the following table.

TABLE Pharmacokinetic parameters for melphalan in whole blood and plasma following intravenous administration to male Sprague Pawley rats at a nominal dose of 2.0 mg/kg with a formulation containing a cyclodextrin derivative (SBE_(6.5)-β-CD) and a formulation free from a cyclodextrin derivative (“CD-free formulation”). Whole-Blood. Plasma SBE.6.5-1•CP CD-free SBE_(6.5)-β-CD CD-free Melphalan (27% w/v) formulation (27% w/v) formulation Apparent t_(1/2) (h) 0.8 ± 0.1  0.9 ± 0.1 0.8 ± 0.1 0.9 ± 0.1 CL_(total) (mL/min/kg) 10.9 ± 2.0  12.2 ± 1.3 8.1 ± 1.1 9.3 ± 1.4 V_(z) (L/kg) 0.8 ± 0.2  1.0 ± 02 0.6 ± 0.1 0.7 ± 0.2 AUC_(0-inf)/D 94.2 ± 16.6 82.8 ± 8.8 125.4 ± 15.0  109.6 ± 16.1  (μM · min · kg/μmol)

FIGS. 5A-5B provide graphic representations of the dose-normalized whole blood (FIG. 5A) and plasma (FIG. 5B) concentrations of melphalan following intravenous administration for the SBE_(6.5)-β-CD-containing: ′melphalan formulation (●), n=4, and the cyclodextrin-free (o), n=5, melphalan formulation (ALKERAN® for Injection, GlaxoSmithKline). Referring to FIGS. 5A-5B, the data is presented as mean with error bars indicating a. single standard deviation. Melphalan exhibited bi-exponential pharmacokinetics it both whole blood and plasma and the apparent terminal elimination phase was well-defined within the: 8 hour post-dose blood sampling period, with the apparent terminal elimination half-life. The mean whole blood and plasma concentration versus time profiles for the formulation that: contained SBE_(6.5)-β-CD. (27% w/V) and cyclodextrin-free formulation were essentially super-imposable and there were: no statistically significant differences in any of the pharmacokinetic parameters between the two formulations (p>0.05), Thus, as shown in the above table, -in the rat the in vivo pharmacokinetic parameters for a melphalan formulation containing SBE_(6.5)-β-CD are essentially identical to the pharmacokinetic parameters for a cyclodextrin-free formulation (i.e., ALKERAN® for Injection, GlaxoSmithKline).

Additionally, in both formulations the percentage of the melphalan dose excreted in urine excreted as unchanged compound up to 24 hours post-dose was low: for the cyclodextrin-free formulation the average was 2.7±1.7%, and for the formulation containing SBE_(6.5)-β-CD the average was 2:3±2%.

The data show that pharmacokinetic parameters, including half-life, AUC, volume of distribution, clearance; and the extent of renal elimination of melphalan were essentially unchanged between ALKERAN® for Injection (GlaxoSmithKline) and the melphalan formulation that contained SAE_(6.5)-β-CD/. Specifically, the mean whole blood and plasma concentration versus time profile of melphalan with and without SBE_(6.5)-β-CD are essentially super-imposable. The results demonstrate that SBE_(6.5)-β-CD had no observable difference in, blood or plasma versus time profiles for melphalan in the rat model. Furthermore, there was no apparent difference in the urinary excretion of melphalan: in the rat model.

Example 17

A Phase lla, multicenter, open-label, randomized, efficacy and safety study of melphalan hydrochloride administered by injection using a propylene glycol-free diluent vehicle has been conducted in 3 human multiple myeloma patients who underwent myeloablative conditioning in preparation for autologous transplantation. The study is on-going.

The primary goal of the study was/is to determine the rate of myeloablation and neutrophil engraftment in multiple myeloma patients who receive a high-dose of melphalan hydrochloride via injection in which one dose is administered using a propylene glycol diluent and one: dose is administered using a propylene glycol-free diluent. The administering, is used as myeloablative therapy prior to autologous stem cell transplantation.

The secondary goal of the study is to determine: (a) the rate of platelet engraftment in multiple myeloma patients receiving high-dose melphalan hydrochloride via injection using both a propylene glycol diluent and, a propylene glycol-free diluent prior to autologous: stem cell transplantation; (b) the median, time to engraftment of neutrophil and platelets in multiple myeloma patients receiving high-dose melphalan hydrochloride via: injection, using both a propylene glycol diluent and a propylene glycol-free diluent prior to autologous stem cell transplantation; (c) the response rate (stringent complete response [sCR], complete response [CR], very good partial response [VGPR], partial response [PR], stable disease [SD], or progressive disease [PD]) at autologous stem cell transplantation at day +100 in multiple myeloma patients receiving high-dose melphalan hydrochloride via injection using a propylene glycol diluent and a propylene glycol-free diluent, prior to autologous stem cell transplantation; (d) the toxicity profile of high-dose melphalan hydrochloride via injection using both a propylene glycol diluent and a propylene glycol-free diluent in multiple myeloma patients undergoing autologous stem cell transplantation; (e) the rate of treatment-related mortality during the first 100 days after autologous stem cell transplantation in multiple myeloma patients receiving high-dose melphalan hydrochloride via injection, using a propylene glycol diluent and a propylene glycol-free diluent; and (f) the pharmacokinetics of melphalan hydrochloride via injection using a propylene glycol diluent compared with the pharmacokinetics of melphalan hydrochloride via injection using a propylene glycol-free diluent (i.e., a cyclodextrin derivative) in multiple myeloma patients undergoing autologous stem cell transplantation.

Patients were screened prior to enrollment in the study. Patients from any of the following classes were qualified for inclusion in the study:

Patients with symptomatic multiple myeloma requiring treatment at diagnosis or anytime thereafter;

Patients with multiple myeloma who qualify for autologous stem cell transplantation therapy who have received appropriate primary induction therapy for transplantation;

Patients who are 70 years of age or younger at time of transplantation (patients greater than 70 years of age may qualify on a case-by-case basis if the patient meets criterion based on institution's standard of practice);

Patients with an adequate autologous graft, defined as an un-manipulated, cryopreserved, peripheral, blood stern cell or bone marrow stem cell graft containing at least 2×10⁶ CD34+ cells/kg based upon patient weight, along with a reserve of 2×10⁶ CD34+ cells/kg that is stored in a separate bag; and

Patients with adequate organ function as Measured by:

-   -   Cardiac: Left ventricular ejection fraction at rest >40%;     -   Hepatic: Bilirubin <2× the upper limit of normal, and ALT/AST         <3×ULN;     -   Renal: -Creatinine clearance >40 mL/minute; and     -   Pulmonary: DLCQ, FEV₁, PVC. >50% of predicted value (corrected         for Hgb) or O₂ saturation >92% on room air.

All patients have received antiemetics hydration, and infection prophylaxis according to -institutional guidelines. Patients, followed institutional guidelines regarding hospitalization. Patients returned for daily laboratory tests (CBC with differential and platelets and a basic chemistry panel) until neutrophil engraftment, and then returned for weekly safety evaluations until autologous stem cell transplantation Day +30. The following safety, efficacy, and pharmacokinetic evaluations were performed prior to the first: dose of melphalan, and at the following post-dose time points:

-   -   Twelve blood samples were taken at specific time points for a         pharmacokinetic evaluation. Blood samples were collected         immediately prior to and after receiving the melphalan dose;     -   Vital signs were recorded hourly′ during the first: eight hours         after receiving each dose of melphalan, then repeated once:         daily until hospital. discharge, and then weekly until Day +30.         Weight were collected at hospital discharge and at Day +30;     -   A 12-lead electrocardiograph, (ECG) along with a 10 to 20 second         rhythm strip was collected twice weekly until hospital         discharge, then a 12-lead ECG (without a rhythm strip), was         collected weekly until Day +30;     -   A focused physical examination was performed daily until         hospital discharge, them a complete physical examination was         performed weekly until Day +30;     -   Toxicity grading and evaluation for AEs/SAEs was according to         NC1-CTC AE Version 3.0 during the entire Study Period;     -   Complete blood count with differential and platelet count was         performed daily until neutrophil and platelet engraftment, -then         weekly until Day +30;     -   Eastern Cooperative Group performance status was examined at the         time of hospital discharge, then weekly until Day +30;     -   Basic serum chemistry panel (sodium, potassium, chloride;         glucose; creatinine, bicarbonate, and BUN) daily until         neutrophil engraftment;     -   Full serum chemistry panel (sodium, potassium, chloride,         magnesium, bicarbonate, glucose, total protein, albumin,         calciiim1, phosphate, uric acid, BUN, creatinine, CPK, total         bilirubin, alkaline phosphate, LDH, SGOT, and SOFT) will be         monitored weekly until Day +30;     -   Urinalysis (specific, gravity, pH, protein, glucose, ketones,         nitrite, IU3Cs, and WBCs) was monitored twice weekly until         hospital discharge, then weekly until Day ±30; and     -   Concomitant medications was recorded during the entire study         period.

A melphalan dose of 200 g/m² was divided into two separate, consecutive doses of 100 mg/m² administered on two separate days (Day −3 and Day −2) prior to the patients receiving an autologous stem cell transplantation. For the calculation of body surface area, actual body weight was used for patients who weighed less than or between 100% to 130% of their ideal body weight. Patients who weighed more than 130% of their ideal body weight were dosed based on a body surface area obtained by calculating the patient's adjusted body weight.

Patients were randomly chosen to receive, the first melphalan Idose of 100 mg/m² (on Day −3) via either a composition comprising a propylene glycol diluent (i.e., Melphalan IIC1 injectable, Bioniche Pharma USA) or a composition comprising, a cyclodextrin derivative (SBE_(6.5)-β-CD, CAPTISOL®, at a concentration of 125 mM). Patients who randomly received the first melphalan dose of 100 mg/m² as a composition comprising a cyclodextrin derivative (on Day −3) then received a second melphalan dose of 100 mg/m² (On Day −2) using the composition comprising a: propylene glycol diluent. Conversely, patients: who randomly received the first melphalan dose of 100 mg/m² as a composition comprising a propylene glycol diluent (on Day −3) then received a second melphalan dose of 100 mg/m² (on Day −2) using the composition comprising a cyclodextrin derivative.

For the composition comprising a cyclodextrin derivative, a dry powder composition containing melphalan as a hydrochloride salt was diluted with normal saline to a melphalan concentration: no greater than 0.45 mg/mL and a cyclodextrin concentration of 125 mM. The diluted solution was infused over 60 minutes via a central voicing catheter.

The composition comprising a propylene glycol diluent was administered using a cyclodextrin-free composition (Melphalan HCl Injectable, Bioniche Pharma USA) using the protocol described herein supra.

Following one day of rest after the myeloablative conditioning (Day −1). Patients received an autologous graft with a minimum cell dose of 2×10⁶ CD34⁺ cells/kg of patient; body weight (Day 0). Cryopreservation and thawing of: the product was consistent with. Foundation for the Accreditation of Cellular Therapy standards and local institutional practice. The graft was infused per institutional, protocol. Starting on. Day +5, G-CSF was be administered at a dose of 5 ug/kg/day until absolute neutrophil count Was greater than 500/mm³:

Blood Samples for pharmacokinetic evaluation a melphalan were collected after each dose of melphalan and the pharmacokinetic parameters for in vivo melphalan distribution were evaluated. Samples for evaluation of the pharmacokinetic parameters were collected by taking 5 MT-venous blood samples immediately prior to melphalan administration and at 0, 10, 20, 30, 60, 90, 120; 180, 240, 360; and 480 minutes following the end of the melphalan infusion. Pharmacokinetic parameters were determined by nonparametric pharmacokinetic data analysis: techniques: Pharmacokinetic parameters computed from plasma drug: concentration.-time data include the following;

-   -   C_(max), derived from the individual raw data;     -   T_(max) derived from the individual raw data;     -   Apparent terminal first-order elimination rate constant         (k_(el));     -   Apparent elimination t_(1/25);     -   Area under the plasma concentration-time curve to the last         measurable time point (AUC₀₋₁), calculated by the trapezoidal         rule; and     -   Area under the plasma concentration-time curve from the last         measurable time point extrapolated to infinity (AUC_(t-∞)),         determined from the concentration at the last measurable time         point divided by the k_(el).

The plasma concentrations and pharmacokinetic ′parameters were summarized using descriptive statistics, The data from patients 1-3 is shown in the following table.

TABLE Individual Patient: melphalan pharmacokinetic: parameters after intravenous administration of a. melphalan formulation, that contained SBE_(6.5)-β-CD and a cyclodextrin-free melphalan. formulation (i.e., Melphalan HCI Injectable, Bioniche Phamta USA). SBF.45.57-13-CD CD-free Patient Parameter (27% w/v) formulation Ratio 1^(a) C_(max) (ng/mL) 3,230 2,160 1.50 T_(max) (min) 10 20 — AUC_(0-t) 259,073 202,714 1.28 AUC_(0-∞) 264,656 208,028 1.27 λ_(z) (min⁻¹) 0.0105 0.0103 — T_(1/2) (min) 65.8 67.1 — 2^(b) C_(max) (ng/mL) 2,730 2,010 1.36 T_(max) (min) 10 10 — AUC_(0-t) 198,051 151,456 1.31 AUC_(0-∞) 202,728 154,130 1.32 λ_(z) (min⁻¹) 0.0103 0.0113 — T_(1/2) (min) 67.4 61.6 — 3^(a) C_(max) (ng/mL) 4,590 2890 1.59 T_(max) (min) 10 10 — AUC_(0-t) 306,432 230,681 1.33 AUC_(0-∞) 314,108 336,059 1.33 λ_(z) (min⁻¹) 0.0104 0.0104 — T_(1/2) (min) 68.5 66.4 — ^(a)Patients 1 and 3 were administered, the SBE_(6.5)-β-CD-containing formulation on Day −3 and the CD-free formulation on Day. 72. ^(b)Patient 2 was administered the CD-free formulation on Day −3 and the SBE_(6.5)-β-CD - containing formulation on Day −2.

FIG. 6, provides a graphic representation of the wean plasma melphalan concentration in a human patient after intravenous administration of a melphalan formulation containing a cyclodextrin derivative (SBE_(6.5)-β-CD) and after intravenous administration of a cyclodextrin-free rnelphalan formulation: (Melphalan HCl Injectable, Bioniche Pharrna USA). Referring to FIG. 6 and the data in the above table; the in vivo distribution of melphalan administered with a sulfoalkyl ether cyclodextrin derivative provides a nearly 50% increase the maximum in vivo concentration of melphalan, and approximately a 30% increase, in the area under the plasma conde-titration curves (i.e., for both AUC_(0-t) and AUC_(0-∞)). As shown in. the table above, the data for patients 2 and 3 exhibited similar pharmacokinetic results. In view of pharmacokinetic data obtained for these melphalan formulations in: the rat model the enhancement in C_(max) and AUC for the SBE_(6.5)-β-CD-containing melphalan formulation in human patients is wholly unexpected.

As noted above, the study is on-going. The primary efficacy end points, which will be based on an intent-to-treat analysis of all patients; will be the rate of myeloablation and the rate neutrophil engraftment. The following definitions will be used for these endpoints:

Myeloablation will be defined as any: of the following:

-   -   Absolute neutrophil count less than 0.5×10⁹/L;     -   Absolute lymphocyte count less than. 0.1×10⁹/L or     -   Platelet count less than 20,000/mm³ or bleeding requiring         transfusion.

The first of two consecutive days for which cell counts drop below these cut-off levels, will be recorded as the date of myeloablation,

Neutrophil engraftment is defined as absolute neutrophil count greater than 0.5×10⁹/L on three consecutive, daily assessments.

Secondary efficacy end points will be based on the following criteria:

-   -   The rate of platelet engraftment, which will be defined as an         un-transfused platelet measurement >20,000/mm³ on three         consecutive daily assessments;|     -   The time to neutrophil engraftment, which will, be defined as         the first of three assessments where absolute neutrophil count         is greater than 0.5×10⁹/L;|     -   The time to platelet engraftment, which will be defined as the:         first of three consecutive daily assessments where un-transfused         platelet measurement is greater than 20,000/mm³;|     -   The rate of non-engraftment, which will be defined as a failure         to reach an absolute neutrophil count greater than: 0.5×10⁹/L.         on three consecutive: daily assessments by autologous stem cell         transplantation Day +100;|     -   The rate of late graft failure or late rejection, which will be         defined as development of absolute neutrophil: count less than         0.5×10⁹/L after having engrafted within the first 100 days;|     -   The rate of multiple myeloma response (sCR, CR, VGPR, PR, SD, or         PD); which, will be defined according to International: Working         Group: criteria at Day +100; and|     -   The rate of treatment-related mortality, which will be defined         as death without relapse or progression at Day +100.

The clinical trial is expected to demonstrate that melphalan administered with a cyclodextrin derivative (SBE_(6.5)-β-CD) is therapeutically effective and safe for use in subjects for whom, a stem cell transplantation has been indicated as conditioning prior to stem cell transplantation.

Example 18

A Phase IIb, multi-center, open-label, non-randomized, efficacy and safety study of melphalan hydrochloride administered by injection using a propylene glycol-free vehicle will be conducted in human multiple myeloma patients who have symptomatic multiple myeloma and, qualify for ASCT.

The parameters of the study will be similar to those described in′ Example 17, except that all patients will be administered a propylene glycol-free: melphalan composition (100 mg/m²) on Day −3 and Day −2 using a melphalan composition that includes a cyclodextrin derivative. Otherwise, the inclusion criteria, exclusion criteria, safety criteria, dosing, treatment, and efficacy endpoints will be similar to those described above in Example 17.

CONCLUSION

These examples illustrate possible embodiments of the present invention. While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation it will be apparent to persons skilled in the relevant art that various changes in farm and detail can be made therein without departing from the spirit and scope: of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above-described, exemplary embodiments, but should be defined only in accordance with the following claims, and their equivalents.

All documents cited herein, including journal articles or abstracts, published or: corresponding U.S. or foreign patent applications, issued or foreign patent % or any other documents, are each entirely incorporated by reference herein, including all data, tables, figures, and text presented ′in the cited documents. 

What is claimed is:
 1. A method of treating a subject suffering from a neoplastic disorder, the method comprising: diluting a composition with an aqueous diluent to provide a dilute pharmaceutical composition comprising 25 mg to 125 mg of melphalan and a cyclodextrin derivative of formula 1:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are independently —H, a straight-chain or branched C₁-C₁-(alkylene)-SO₃— group, or an optionally substituted straight-chain or branched C₁-C₆ group; wherein at least one of R₁, R₂, P₃, P₄, R₅, R₆, R₇, K₈ and R₉ is a straight-chain or branched C₁-C₈-(alkylene)-SO₃ group; wherein the dilute pharmaceutical composition has, a pH of about 4 to about 6; wherein the cyclodextrin derivative is present in a concentration of at least 50:1 (w/w) relative to: the melphalan, wherein the melphalan in the dilute, pharmaceutical composition degrades by 2% or less at about 25Q C. within 0.5 hours after the diluting; and administering the dilute pharmaceutical composition, by injection to the subject in need thereof.
 2. The method of claim 1, wherein the neoplastic disorder is selected from: myeloma, Multiple myeloma, melanoma, acute myelogenous leukemia, malignant melanoma, breast cancer, ovarian cancer, testicular cancer, advanced prostate cancer, a neuroendocrine cancer, metastatic melanoma, a metastatic neuroendocrine tumor, a metastatic adenocarcinoma tumor, hepatocellular carcinoma, osteogenic sarcoma, polycythemia veraplasma; plasma cell neoplasm, amyloidosis, scleromyxedema, and combinations thereof.
 3. The method of claim 2, wherein the neoplastic disorder is multiple myeloma and the administering is systemic and provides, palliative treatment of the multiple myeloma.
 4. The method of claim 1, wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉. is a hydroxy-substituted-C₃ group.
 5. The method of claim 1, wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are independently a straight-chain or branched C₁-C₈-(alkylene)-SO₃— group having a degree of substitution of 4 to 8 per cyclodextrin derivative, and the remaining substituents are —H.
 6. The method of claim 1, wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is substituted with a straight-chain C₄-(alkylene)-SO₃— group.
 7. The method of claim 1, wherein the cyclodextrin derivative is a compound of formula IL

wherein R═(H)_(21-x), or ((CH₂)₄SO₃Na⁺)_(x) and x=6.0-7.1; wherein the pharmaceutical composition comprises about 50 mg of melphalan as a hydrochloride salt; and wherein the cyclodextrin derivative is: present in a concentration, of 50:1 to 100:1 (w/w) relative to the melphalan.
 8. The method of claim 1, wherein, the dilute pharmaceutical composition is substantially free of an alcohol.
 9. The method of claim 1, wherein the aqueous diluent is a saline solution.
 10. The method of claim 1, wherein the, subject suffering from the neoplastic disorder is pediatric subject.
 11. The method of claim 1, wherein the melphalan in the dilute pharmaceutical composition degrades by 4% or less at 25° C. within 10 hours after the diluting:
 12. The method of claim 1, wherein the dilute pharmaceutical composition is stored about 0.5 hours to about 18 hours prior to the administering.
 13. The method of claim 1, wherein the administering, provides a melphalan C_(max) in the subject suffering from a neoplastic disorder that is at least 20% or greater than a melphalan C_(max) provided by a melphalan formulation containing an equivalent dose of melphalan and lacking the cyclodextrin derivative.
 14. The method of claim 1, wherein the administering provides a melphalan AUC_(0-t) in the subject suffering from a neoplastic disorder that is at least 20% or greater than a melphalan AUC_(0-t) provided by a melphalan formulation containing an: equivalent dose of melphalan and lacking the cyclodextrin derivative.
 15. A method for conditioning a subject for whom a stem cell transplantation has been indicated, the method comprising: administering a melphalan dose of 50 mg/m² to 300 mg/m² per day to the subject for whom a stem cell transplantation has been indicated, wherein the melphalan dose is administered in a pharmaceutical: composition comprising melphalan and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃— group, or an optionally substituted straight-chain or branched C₁-C₆ group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branched C1-C8-(alkylene)-SO₃ group; wherein the pharmaceutical composition has a pH of about 4 to about 6; and wherein the cyclodextrin derivative is present in a ratio of at least 25:1 (w/w) relative to the melphalan.
 16. The method of claim 15, wherein the administering is for a period of two or more days.
 17. The method of claim 15, wherein, the subject in need of the stem cell transplantation is a pediatric subject.
 18. The method of claim 15, wherein the administering is performed intravenously.
 19. The method of claim 15, wherein, the administering is performed via a limb perfusion.
 20. The method of claim 15, wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a hydroxy-substituted-C₃ group.
 21. The method of claim 15, wherein the cyclodextrin derivative is a compound of formula II:

wherein R═(H)_(21-x) or (—(CH₂)₄—SO₃—Na⁺)_(x), and x=6.0-7.1; wherein the pharmaceutical composition comprises about 200 mg of melphalan as a hydrochloride salt; and wherein the cyclodextrin derivative is present in a ratio of 25:1. to 35:1 (w/w) relative to the: melphalan.
 22. The method of claim 15, wherein the subject for whom. A stem cell transplantation has: been indicated suffers from a disease or disorder selected from: myeloma, multiple myeloma, a lymphoma, non-Hodgkin lymphoma, leukemia, acute myeloid leukemia, Hodgkin's disease, acute lymphoblastic leukemia, a: myelodysplastic syndrome, a myeloproliferative disorder; chronic myelogenous leukemia, neuroblastoma, aplastic anemia, chronic granulocytic leukemia, a neuroblastoma, sickle-cell disease, osteogenic sarcoma, Ewing's sarcoma, a desmoplastic small round cell tumor, plasma cell neoplasm, amyloidosis, scleromyxedema, and combinations thereof.
 23. The method of claim 15, comprising diluting a concentrated melphalan composition with an aqueous diluent to provide the pharmaceutical composition,
 24. The method of claim 23, wherein the concentrated melphalan composition comprises 50 mg to 500 mg of melphalan.
 25. The method of claim 23, wherein the concentrated melphalan composition comprises about 200 mg.
 26. The method of claim 15, wherein the pharmaceutical composition is substantially free of an alcohol.
 27. The method of claim 23, wherein the aqueous diluent is a saline solution.
 28. The method of claim 23, wherein the melphalan in the pharmaceutical composition degrades by 4% or less at about 25° C. within 10 hours after the diluting.
 29. The method of claim 23, wherein the pharmaceutical composition is stored about 0.5 hours to about 12 hours prior to the administering.
 30. The method of claim 15, wherein the administering provides a melphalan C_(max) in the subject for whom a stem cell transplantation has been indicated that is at least 20% or greater than a melphalan C_(max) provided by a melphalan formulation containing an equivalent dose of melphalan and lacking the cyclodextrin derivative.
 31. The method of claim 15, wherein the administering provides a melphalan AUC_(0-t) in the subject for whom a stem cell transplantation has: been indicated that is at least 20% or greater than a melphalan. AUC_(0-t) provided by a. melphalan formulation containing an equivalent dose of melphalan and lacking the cyclodextrin derivative.
 32. A pharmaceutical composition comprising: 25 mg to 125 mg of melphalan as a hydrochloride salt; an optional buffer; and a cyclodextrin derivative of formula 1:

wherein n s 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group, or an optionally substituted straight-chain or branched C₁-C₆ group; wherein at least one of R1, R2, R3, R4, R5, R6, R7, R8 and R9 is a, straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein the pharmaceutical composition has a pH of about 4 to about 6, wherein dilution of the pharmaceutical composition with an aqueous solution provides a dilute pharmaceutical composition in which the melphalan degrades by 2% or less at about 25° C. within 5 hours after the dilution; and wherein the cyclodextrin derivative is present in a ratio of 50:1 to 100:1 (w/w) relative to the melphalan.
 33. The pharmaceutical composition of claim 32, wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a hydroxy-substituted-C₃ group.
 34. The pharmaceutical composition of claim 32, wherein the cyclodextrin derivative is a compound of formula: II:

wherein R═(H)_(21-x) or (—(CH₂)₄SO₃Na⁺)_(x), and x=6.0-7.1; wherein the pharmaceutical composition comprises about 50 mg of melphalan as a hydrochloride salt; and wherein the cyclodextrin derivative, is present in a ratio, of about 55:1 (w/w) relative to the melphalan.
 35. A pharmaceutical composition comprising; 150 mg to 250 mg of melphalan as a hydrochloride salt; an optional buffer; and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are independently —H, a straight-chain or branched C₁-C₈(alkylene)-SO₃— group, or an optionally substituted straight-chain or branched C₁-C₆ group; wherein at least one of R1, R2, R3, R4, R5, R6, R7, R8 and R9 is a straight-chain or branched C₁-C₈(alkylene)-S0₃ ⁻ group; wherein the pharmaceutical composition has a pH of about 4 to about 6, wherein dilution of the pharmaceutical composition with an aqueous solution provides a melphalan solution in. which the melphalan degrades by 2% or loss a about 25° C. within 5 hours after the dilution; and wherein the cyclodextrin derivative is present in a ratio of 25:1 to 35:1 (w/w) relative to the melphalan.
 36. The pharmaceutical composition of claim 35, wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a hydroxy-substituted C₃ group.
 37. The pharmaceutical composition of claim 35, wherein the cyclodextrin derivative is a compound of formula II:

wherein R═(H)_(21-x) or (—(CH₂)₄SO₃Na⁺)_(x), and x=6.0-7.1; wherein the pharmaceutical composition comprises about 200 mg of melphalan as a hydrochloride salt; and wherein the cyclodextrin derivative is present in a ratio of about 30:1 (w/w) relative to the melphalan.
 38. A pharmaceutical kit comprising: a first container comprising 25 mg to 125 mg of melphalan as a hydrochloride salt and an optional water-soluble polymer; and a second container comprising an aqueous diluent, an optional buffer, and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-S0₃ ⁻ group, or an optionally substituted straight-chain or branched C1-C6 group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branched C₁-C₈(alkylene)-S0₃ ⁻ group; wherein the cyclodextrin derivative is present in the second container in a concentration of at least 50:1 (w/w) relative to the melphalan; and wherein combining the first container and. the second. container provides a dilute pharmaceutical composition having a pH of about 4 to about 6 that degrades by 2% or less at about 25° C. within 5 hours after the diluting.
 39. The pharmaceutical kit of claim 38, wherein the first container comprises povidone in an amount of 10 mg to 3.0 mg, and the second container comprises a pH-adjusting agent in a concentration sufficient to provide a pH of about 4 to about 6 when the first container and the second container are combined.
 40. A pharmaceutical kit comprising; a first container comprising 150 mg to 250 mg of melphalan as a hydrochloride salt and. an optional water-soluble polymer; and a second container comprising an aqueous diluent, an optional buffer, and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are independently —H, a straight-chain or branched C₁-C₈(alkylene)-S0₃ group, or an optionally substituted straight-chain or branched C₁-C₆ group; wherein at least one of RI, R2, R3, R4, R5, R6, R7, R8 and R9 is a straight-chain or branched C₁-C₈(alkylene)-S0₃ ⁻ group; wherein the cyclodextrin derivative is present in the second container in a concentration of 25:1 to 35:1 (w/w) relative to the melphalan; and wherein combining the first container and the second container provides a dilute pharmaceutical composition having a pH of about 4 to about 6 that degrades by 2% or less at about 25° C. within 5 hours after the diluting.
 41. The pharmaceutical kit of claim 40, wherein the first container comprises povidone in an amount of 10 mg to 30 mg, and the second container comprises a pH-adjusting agent in a concentration sufficient to provide a pH of about 4 to about 6 when the first container and the second container are combined.
 42. The pharmaceutical kits of claim 40, wherein the cyclodextrin derivative is a compound of formula II:

wherein R═(H)_(21-x) or (—(CH₂)₄SO₃ ⁻Na⁺)_(x), and x=6.0-7.1; wherein the first container comprises about 200 mg of melphalan as a hydrochloride salt; and wherein the cyclodextrin derivative is present, in the second container in an amount of about 30:1 (w/w) relative to the melphalan. 